Amyloid beta detection by mass spectrometry

ABSTRACT

Provided are methods for the detection or quantitation of amyloid beta. In a particular aspect, provided herein are methods for detecting amyloid beta or fragments thereof by mass spectrometry. In another aspect, provided herein are methods for determining the ratio of amyloid beta 42 (Aβ42) to amyloid beta 40 (Aβ40). In another aspect, provided herein are methods for diagnosis or prognosis of Alzheimer&#39;s disease or dementia.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application in a continuation application of U.S. patentapplication Ser. No. 15/277,879, filed Sep. 27, 2016, now U.S. Pat. No.11,360,098, which claims benefit of U.S. Provisional Application No.62/234,027, filed Sep. 28, 2015, and U.S. Provisional Application No.62/277,772, filed Jan. 12, 2016, each of which is incorporated byreference herein in its entirety.

FIELD OF THE INVENTION

The invention relates to the detection or quantitation of amyloid beta.In a particular aspect, the invention relates to methods for detectingamyloid beta or fragments thereof by mass spectrometry.

BACKGROUND OF THE INVENTION

Alzheimer's disease is the most common form of dementia affecting theelderly population. Alzheimer's disease is characterized by aprogressive decay of cognitive abilities, in particular, memory andlearning. One of the hallmarks of the disease is neuritic plaquescomposed of amyloid beta (Aβ or Abeta) peptides.

The accuracy and sensitivity of current clinical diagnostic methods topredict or diagnose Alzheimer's disease is low Immunoassays arecurrently offered to detect amyloid beta, which is a biomarkerpredictive of progression to Alzheimer's disease. However,inter-laboratory variations in the results observed with currentlyavailable immunoassays are of concern.

An accurate and sensitive assay for detecting amyloid beta is needed.

SUMMARY OF THE INVENTION

Provided herein are methods for detecting or determining the amount ofamyloid beta (Aβ) in a sample by mass spectrometry, including tandemmass spectrometry.

In certain embodiments, the methods provided herein for determining theamount of amyloid beta comprises (a) purifying amyloid beta in thesample; (b) ionizing amyloid beta in the sample; and (c) determining theamount of the amyloid beta ion(s) by mass spectrometry; wherein theamount of the amyloid beta ion(s) is related to the amount of amyloidbeta in the sample.

In certain embodiments, the methods provided herein for determining theamount of amyloid beta comprises (a) purifying amyloid beta in thesample; (b) ionizing amyloid beta in the sample to produce a precursorion of amyloid beta; (c) generating one or more fragment ions of amyloidbeta; and (d) determining the amount of the ion(s) from step (c) or (d)or both by mass spectrometry; wherein the amount of the amyloid betaion(s) is related to the amount of amyloid beta in the sample.

In certain embodiments, the methods provided herein for determining theamount of amyloid beta comprises (a) digesting amyloid beta in thesample to generate one or more fragments of amyloid beta; (b) purifyingthe one or more amyloid beta fragments; (c) ionizing the one or moreamyloid beta fragments to produce a precursor ion; (d) generating one ormore fragment ions; and (e) determining the amount of the ion(s) fromstep (c) or (d) or both by mass spectrometry; wherein the amount of theion(s) is related to the amount of amyloid beta fragment(s) in thesample.

In certain embodiments, provided herein are methods for determining theamount of amyloid beta 42 (Aβ42). In some embodiments, the Aβ42 fragmentcomprises the sequence GAIIGLMVGGVVIA (SEQ ID NO:4). In someembodiments, the methods comprise (a) digesting amyloid beta in thesample to generate amyloid beta 42 (Aβ42); (b) purifying Aβ42; (c)ionizing Aβ42 to produce a precursor ion; (d) generating one or morefragment ions of Aβ42; and (e) determining the amount of the ion(s) fromstep (c) or (d) or both by mass spectrometry; wherein the amount of theion(s) is related to the amount of Aβ42 in the sample.

In certain embodiments, provided herein are methods for determining theamount of amyloid beta 40 (Aβ40). In some embodiments, the Aβ40 fragmentcomprises the sequence GAIIGLMVGGVV (SEQ ID NO:2). In some embodiments,the methods comprise (a) digesting amyloid beta in the sample togenerate amyloid beta 40 (Aβ40); (b) purifying Aβ40; (c) ionizing Aβ40to produce a precursor ion; (d) generating one or more fragment ions ofAβ40; and (e) determining the amount of the ion(s) from step (c) or (d)or both by mass spectrometry; wherein the amount of the ion(s) isrelated to the amount of Aβ40 in the sample.

In certain embodiments, provided herein are methods for determining theamount of amyloid beta 42 (Aβ42) and amyloid beta 40 (Aβ40). In someembodiments, the methods comprise (a) digesting amyloid beta in thesample to generate amyloid beta 42 (Aβ42) and amyloid beta 40 (Aβ40);(b) purifying Aβ42 and Aβ40; (c) ionizing Aβ42 and Aβ40 to produceprecursor ions; (d) generating one or more fragment ions of Aβ42 andAβ40; and (e) determining the amount of the ion(s) from step (c) or (d)or both by mass spectrometry; wherein the amount of the ion(s) isrelated to the amount of Aβ42 and Aβ40 in the sample.

In certain embodiments, provided herein are methods for determining theratio of amyloid beta 42 (Aβ42) to amyloid beta 40 (Aβ40). In someembodiments, the methods comprise (a) digesting amyloid beta in thesample to generate amyloid beta 42 (Aβ42) and amyloid beta 40 (Aβ40);(b) purifying Aβ42 and Aβ40; (c) ionizing Aβ42 and Aβ40 to produceprecursor ions; (d) generating one or more fragment ions of Aβ42 andAβ40; and (e) determining the amount of the ion(s) from step (c) or (d)or both by mass spectrometry; and (f) determining the ratio of Aβ42 toAβ40. In some embodiments, the methods comprise determining the ratio ofAβ40 to Aβ42.

In certain embodiments, provided herein are methods for diagnosis orprognosis of Alzheimer's disease or dementia, the method comprisingdetermining the amount of amyloid beta in a test sample by massspectrometry; wherein an abnormal levels of amyloid beta is predictiveor diagnostic of Alzheimer's disease. In some embodiments, the methodsmay include: (a) purifying amyloid beta in the sample; (b) ionizingamyloid beta in the sample; and (c) determining the amount of theamyloid beta ion(s) by mass spectrometry; and (d) the amount of theamyloid beta ion(s) is related to the amount of amyloid beta in thesample; wherein the abnormal levels of amyloid beta is predictive ordiagnostic of Alzheimer's disease. In some embodiments, the methodscomprise determining the ratio of amyloid beta fragments. In someembodiments, the methods comprise determining the ratio of amyloid beta42 (Aβ42) to amyloid beta 40 (Aβ40). In some embodiments, the methodscomprise determining the ratio of amyloid beta 42 (Aβ40) to amyloid beta40 (Aβ42).

In certain embodiments, the methods provided herein comprise pretreatingsurfaces of equipment that come in contact with the sample. In someembodiments, the pretreatment comprises pre-coating the surfaces ofequipment with an agent that prevents amyloid beta or fragments thereoffrom sticking to the surfaces. In some embodiments, the pretreatmentcomprises bacterial lysate pretreatment. In some embodiments, thepretreatment comprises E. coli lysate pretreatment. In some embodiments,the E. coli lysate comprises a trypsin-digested E. coli lysate. In someembodiments, the pretreated equipment includes, but not limited to, testtubes or plates, pipette tips, sample preparation apparatus, liquidchromatography apparatus, and mass spectrometry apparatus.

In certain embodiments, the methods provided herein comprise treating orincubating the sample with an agent that stabilizes amyloid beta orfragments thereof. In some embodiments, the methods provided hereincomprise treating or incubating the sample with an amyloid betaantibody. In some embodiments, the methods provided herein comprisetreating or incubating the sample with at least two distinct amyloidbeta antibodies. In some embodiments, the amyloid beta antibodycomprises an antibody that binds to the C-terminus of amyloid beta. Insome embodiments, the amyloid beta antibody comprises an antibody thatbinds to the N-terminus of amyloid beta. In some embodiments, the agentthat stabilizes amyloid beta comprises an apolipoprotein. In someembodiments, the agent that stabilizes amyloid beta comprisesapolipoprotein E2. In some embodiments, the agent that stabilizesamyloid beta comprises apolipoprotein E4. In some embodiments, the agentthat stabilizes amyloid beta comprises an antibody that binds to theC-terminus of amyloid beta, an antibody that binds to the N-terminus ofamyloid beta, apolipoprotein E2, apolipoprotein E4, or a combinationthereof. In some embodiments, the agent that stabilizes amyloid betaprovided herein confers stability for at least 1 month at −70° C. Insome embodiments, the agent that stabilizes amyloid beta provided hereinconfers stability for at least 2 months at −70° C. In some embodiments,the agent that stabilizes amyloid beta provided herein confers stabilityfor at least 3 months at −70° C. In some embodiments, the agent thatstabilizes amyloid beta provided herein confers stability through afreeze-thaw cycle. In some embodiments, the agent that stabilizesamyloid beta provided herein confers stability through at least twofreeze-thaw cycles. In some embodiments, the agent that stabilizesamyloid beta provided herein confers stability through at least threefreeze-thaw cycles. In some embodiments, the agent that stabilizesamyloid beta provided herein confers stability through at least fourfreeze-thaw cycles.

In certain embodiments, the methods provided herein comprise digestingamyloid beta in the sample. In some embodiments, the methods providedherein comprise digesting amyloid beta with an enzyme. In someembodiments, the enzyme is Lys-C. In some embodiments, the methodsprovided herein comprise digesting amyloid beta with urea. In someembodiments, the urea is in a concentration suitable for proteindigestion. In some embodiments, the urea is 6M urea. In someembodiments, the methods provided herein comprise digesting amyloid betawith urea and Lys-C. In some embodiments, the digestion comprisesdigesting in conditions that reduce digestion time or increase digestionefficiency. In some embodiments, the digestion comprises digesting inmicrowave. In some embodiments, the methods provided herein comprisedetermining the amount of digested amyloid beta.

In certain embodiments, the methods provided herein comprise anextraction. In some embodiments, the methods provided herein comprise amixed mode anion exchange extraction. In some embodiments, the methodsprovided herein comprise a solid phase extraction.

In certain embodiments, the methods provided herein comprise eluting anddrying the sample using heated nitrogen. In some embodiments, the sampleis resuspended in a reconstitution buffer.

In certain embodiments, the purifying the sample comprises a liquidchromatography. In some embodiments, liquid chromatography includes, butnot limited to, reverse phase liquid chromatography (RPLC), highperformance liquid chromatography (HPLC), and high turbulence liquidchromatography (HTLC). In a preferred embodiment, liquid chromatographycomprises HPLC. In some embodiments, HPLC column typically includes amedium (i.e., a packing material) to facilitate separation of chemicalmoieties (i.e., fractionation). Suitable columns may include C-4, C-8,C-12, or C-18 columns. In a preferred embodiment, a suitable HPLC columnis C-4 column.

In certain embodiments, the methods provided herein comprise usingequipment that reduces sticking of amyloid beta to the surfaces ofequipment. In some embodiments, the equipment comprises PEEK (poly etherether ketone) tubing or apparatus. In some embodiments, the equipmentcomprises metal tubing or apparatus.

In certain embodiments, the methods provided herein comprise tandem massspectrometry. In some embodiments, the methods provided herein compriseionizing in positive mode. In some embodiments, the methods providedherein comprise ionizing in negative mode. In some embodiments, themethods provided herein comprise ionizing using heated electrosprayionization (HESI). In some embodiments, the methods provided hereincomprise ionizing using electrospray ionization (ESI). In someembodiments, the methods provided herein comprise ionizing usingatmospheric pressure chemical ionization (APCI). In a preferredembodiment, the methods provided herein comprise ionizing using heatedelectrospray ionization (HESI) in positive mode. In some embodiments,the collision energy is between 5V to 60V. In some embodiments, thecollision energy is between 10V to 50V. In some embodiments, thecollision energy is between 20V to 50V. In some embodiments, thecollision energy is between 20V to 45V.

In certain embodiments, the methods provided herein comprise detectingor determining the amount of amyloid beta 40 (Aβ40). In someembodiments, Aβ40 comprises the sequenceDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV (SEQ ID NO:1). In someembodiments, the methods provided herein comprise detecting ordetermining the amount of a fragment of Aβ40. In some embodiments, theAβ40 fragment comprises the sequence GAIIGLMVGGVV (SEQ ID NO:2). In someembodiments, the Aβ40 fragment comprises a sequence containing anN-terminal or C-terminal winged peptide. In some embodiments, the Aβ40fragment comprises SEQ ID NO:2 and an N-terminal or C-terminal wingedpeptide. In some embodiments, the winged peptide is hydrophilic. In someembodiments, the winged peptide comprises at least one amino acid. Insome embodiments, the winged peptide comprises at least two amino acids.In some embodiments, the winged peptide comprises at least three aminoacids. In some embodiments, the winged peptide comprises at least fouramino acids. In some embodiments, the winged peptide comprises at leastfive amino acids. In some embodiments, the winged peptide comprises atleast six amino acids. In some embodiments, the amount of the Aβ40fragment correlates to the amount of Aβ40 in the sample.

In certain embodiments, the methods provided herein comprise detectingor determining the amount of amyloid beta 42 (Aβ42). In someembodiments, Aβ42 comprises the sequenceDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA (SEQ ID NO:3). In someembodiments, the methods provided herein comprise detecting ordetermining the amount of a fragment of Aβ42. In some embodiments, theAβ42 fragment comprises the sequence GAIIGLMVGGVVIA (SEQ ID NO:4). Insome embodiments, the Aβ42 fragment comprises a sequence containing anN-terminal or C-terminal winged peptide. In some embodiments, the Aβ42fragment comprises SEQ ID NO:4 and an N-terminal or C-terminal wingedpeptide. In some embodiments, the winged peptide is hydrophilic. In someembodiments, the winged peptide comprises at least one amino acid. Insome embodiments, the winged peptide comprises at least two amino acids.In some embodiments, the winged peptide comprises at least three aminoacids. In some embodiments, the winged peptide comprises at least fouramino acids. In some embodiments, the winged peptide comprises at leastfive amino acids. In some embodiments, the winged peptide comprises atleast six amino acids. In some embodiments, the amount of the Aβ42fragment correlates to the amount of Aβ42 in the sample.

In certain embodiments, the methods provided herein comprise detectingor determining the ratio of Aβ40 to Aβ42 (Aβ40:Aβ42). In someembodiments, the methods provided herein comprise detecting ordetermining the ratio of the Aβ40 fragment to the Aβ42 fragment (Aβ40fragment:Aβ42 fragment). In some embodiments, the methods providedherein comprise detecting or determining the ratio of Aβ42 to Aβ40(Aβ42:Aβ40). In some embodiments, the methods provided herein comprisedetecting or determining the ratio of the Aβ42 fragment to the Aβ40fragment (Aβ42 fragment:Aβ40 fragment).

In certain embodiments, the ratio of Aβ42 to Aβ40, or the ratio of theAβ42 fragment to the Aβ40 fragment, of 0.6 or less is predictive ordiagnostic of Alzheimer's disease. In some embodiments, the ratio ofAβ42 to Aβ40, or the ratio of the Aβ42 fragment to the Aβ40 fragment, of0.5 or less is predictive or diagnostic of Alzheimer's disease. In someembodiments, the ratio of Aβ42 to Aβ40, or the ratio of the Aβ42fragment to the Aβ40 fragment, of 0.45 or less is predictive ordiagnostic of Alzheimer's disease. In some embodiments, the ratio ofAβ42 to Aβ40, or the ratio of the Aβ42 fragment to the Aβ40 fragment, of0.4 or less is predictive or diagnostic of Alzheimer's disease. In someembodiments, the ratio of Aβ42 to Aβ40, or the ratio of the Aβ42fragment to the Aβ40 fragment, of 0.35 or less is predictive ordiagnostic of Alzheimer's disease. In some embodiments, the ratio ofAβ42 to Aβ40, or the ratio of the Aβ42 fragment to the Aβ40 fragment, of0.3 or less is predictive or diagnostic of Alzheimer's disease. In someembodiments, the ratio of Aβ42 to Aβ40, or the ratio of the Aβ42fragment to the Aβ40 fragment, of 0.25 or less is predictive ordiagnostic of Alzheimer's disease. In some embodiments, the ratio ofAβ42 to Aβ40, or the ratio of the Aβ42 fragment to the Aβ40 fragment, of0.2 or less is predictive or diagnostic of Alzheimer's disease. In someembodiments, the ratio of Aβ42 to Aβ40, or the ratio of the Aβ42fragment to the Aβ40 fragment, of 0.15 or less is predictive ordiagnostic of Alzheimer's disease.

In certain embodiments, the methods include generating one or moreprecursor ions of Aβ or a fragment thereof. In some embodiments, atleast one of the precursor ions has a mass/charge ratio of 1085.6±0.5 or1269.7±0.5. In some embodiments, the methods may include generating oneor more fragment ions of Aβ or a fragment thereof. In some embodiments,at least one of the fragment ions has a mass/charge ratio of 812.37±0.5,869.4+0.5, 968.43±0.5, 869.39±0.5, 968.44±0.5, 1067.5±0.5, or1180.57±0.5.

In certain embodiments, the methods provided herein comprise adding aninternal standard. In some embodiments, the internal standard comprisesan isotopically labeled internal standard. In some embodiments, theinternal standard comprises ¹³C¹⁵N labeling. In some embodiments, theinternal standard comprises at least one Phe, Leu, or Met labeled with¹³C¹⁵N. In some embodiments, at least one of the precursor ions of theinternal standard has a mass/charge ratio of 1110.7±0.5. In someembodiments, the methods may include generating one or more fragmentions of the internal standard. In some embodiments, at least one of thefragment ions has a mass/charge ratio of 768.48±0.5, 825.5±0.5, or882.52±0.5.

In certain embodiments, the limit of quantitation of the methods is lessthan or equal to 10 ng/mL. In some embodiments, the limit ofquantitation of the methods is less than or equal to 5 ng/mL. In someembodiments, the limit of quantitation of the methods is less than orequal to 4 ng/mL. In some embodiments, the limit of quantitation of themethods is less than or equal to 3 ng/mL. In some embodiments, the limitof quantitation of the methods is less than or equal to 2 ng/mL. In someembodiments, the limit of quantitation of the methods is less than orequal to 1 ng/mL. In some embodiments, the limit of quantitation of themethods is less than or equal to 0.5 ng/mL. In some embodiments, thelimit of quantitation of the methods is less than or equal to 0.2 ng/mL.In some embodiments, the limit of quantitation of the methods is lessthan or equal to 0.1 ng/mL.

In some embodiments, the limit of detection of the methods is less thanor equal to 5 ng/mL. In some embodiments, the limit of detection of themethods is less than or equal to 1 ng/mL. In some embodiments, the limitof detection of the methods is less than or equal to 0.5 ng/mL. In someembodiments, the limit of detection of the methods is less than or equalto 0.1 ng/mL. In some embodiments, the limit of detection of the methodsis less than or equal to 0.05 ng/mL. In some embodiments, the limit ofdetection of the methods is less than or equal to 0.01 ng/mL.

In some embodiments, amyloid beta is not derivatized prior to massspectrometry. In some embodiments, amyloid beta is derivatized prior tomass spectrometry.

In certain embodiments, the sample is a body fluid. In some embodiments,the sample is cerebrospinal fluid (CSF). In some embodiments, the sampleis plasma or serum. In some embodiments, the sample is whole blood. Insome embodiments, the sample is saliva or urine.

In some embodiments, the methods may include adding an agent to thesample in an amount sufficient to deproteinate the sample.

As used herein, unless otherwise stated, the singular forms “a,” “an,”and “the” include plural reference. Thus, for example, a reference to “aprotein” includes a plurality of protein molecules.

As used herein, the term “purification” or “purifying” does not refer toremoving all materials from the sample other than the analyte(s) ofinterest. Instead, purification refers to a procedure that enriches theamount of one or more analytes of interest relative to other componentsin the sample that may interfere with detection of the analyte ofinterest. Samples are purified herein by various means to allow removalof one or more interfering substances, e.g., one or more substances thatwould interfere with the detection of selected amyloid beta parent anddaughter ions by mass spectrometry.

As used herein, the term “test sample” refers to any sample that maycontain amyloid beta. As used herein, the term “body fluid” means anyfluid that can be isolated from the body of an individual. For example,“body fluid” may include blood, plasma, serum, bile, saliva, urine,tears, perspiration, and the like.

As used herein, the term “derivatizing” means reacting two molecules toform a new molecule. Derivatizing agents may include isothiocyanategroups, dinitro-fluorophenyl groups, nitrophenoxycarbonyl groups, and/orphthalaldehyde groups, and the like.

As used herein, the term “chromatography” refers to a process in which achemical mixture carried by a liquid or gas is separated into componentsas a result of differential distribution of the chemical entities asthey flow around or over a stationary liquid or solid phase.

As used herein, the term “liquid chromatography” or “LC” means a processof selective retardation of one or more components of a fluid solutionas the fluid uniformly percolates through a column of a finely dividedsubstance, or through capillary passageways. The retardation resultsfrom the distribution of the components of the mixture between one ormore stationary phases and the bulk fluid, (i.e., mobile phase), as thisfluid moves relative to the stationary phase(s). Examples of “liquidchromatography” include reverse phase liquid chromatography (RPLC), highperformance liquid chromatography (HPLC), and high turbulence liquidchromatography (HTLC).

As used herein, the term “high performance liquid chromatography” or“HPLC” refers to liquid chromatography in which the degree of separationis increased by forcing the mobile phase under pressure through astationary phase, typically a densely packed column

As used herein, the term “high turbulence liquid chromatography” or“HTLC” refers to a form of chromatography that utilizes turbulent flowof the material being assayed through the column packing as the basisfor performing the separation. HTLC has been applied in the preparationof samples containing two unnamed drugs prior to analysis by massspectrometry. See, e.g., Zimmer et al., J. Chromatogr. A 854: 23-35(1999); see also, U.S. Pat. Nos. 5,968,367, 5,919,368, 5,795,469, and5,772,874, which further explain HTLC. Persons of ordinary skill in theart understand “turbulent flow”. When fluid flows slowly and smoothly,the flow is called “laminar flow”. For example, fluid moving through anHPLC column at low flow rates is laminar In laminar flow the motion ofthe particles of fluid is orderly with particles moving generally instraight lines. At faster velocities, the inertia of the water overcomesfluid frictional forces and turbulent flow results. Fluid not in contactwith the irregular boundary “outruns” that which is slowed by frictionor deflected by an uneven surface. When a fluid is flowing turbulently,it flows in eddies and whirls (or vortices), with more “drag” than whenthe flow is laminar Many references are available for assisting indetermining when fluid flow is laminar or turbulent (e.g., TurbulentFlow Analysis: Measurement and Prediction, P. S. Bernard & J. M.Wallace, John Wiley & Sons, Inc., (2000); An Introduction to TurbulentFlow, Jean Mathieu & Julian Scott, Cambridge University Press (2001)).

As used herein, the term “gas chromatography” or “GC” refers tochromatography in which the sample mixture is vaporized and injectedinto a stream of carrier gas (as nitrogen or helium) moving through acolumn containing a stationary phase composed of a liquid or aparticulate solid and is separated into its component compoundsaccording to the affinity of the compounds for the stationary phase.

As used herein, the term “large particle column” or “extraction column”refers to a chromatography column containing an average particlediameter greater than about 35 μm. As used in this context, the term“about” means±10%. In a preferred embodiment the column containsparticles of about 60 μm in diameter.

As used herein, the term “analytical column” refers to a chromatographycolumn having sufficient chromatographic plates to effect a separationof materials in a sample that elute from the column sufficient to allowa determination of the presence or amount of an analyte. Such columnsare often distinguished from “extraction columns”, which have thegeneral purpose of separating or extracting retained material fromnon-retained materials in order to obtain a purified sample for furtheranalysis. As used in this context, the term “about” means±10%. In apreferred embodiment the analytical column contains particles of about 4μm in diameter.

As used herein, the term “on-line” or “inline”, for example as used in“on-line automated fashion” or “on-line extraction” refers to aprocedure performed without the need for operator intervention. Incontrast, the term “off-line” as used herein refers to a procedurerequiring manual intervention of an operator. Thus, if samples aresubjected to precipitation, and the supernatants are then manuallyloaded into an autos ampler, the precipitation and loading steps areoff-line from the subsequent steps. In various embodiments of themethods, one or more steps may be performed in an on-line automatedfashion.

As used herein, the term “mass spectrometry” or “MS” refers to ananalytical technique to identify compounds by their mass. MS refers tomethods of filtering, detecting, and measuring ions based on theirmass-to-charge ratio, or “m/z”. MS technology generally includes (1)ionizing the compounds to form charged compounds; and (2) detecting themolecular weight of the charged compounds and calculating amass-to-charge ratio. The compounds may be ionized and detected by anysuitable means. A “mass spectrometer” generally includes an ionizer andan ion detector. In general, one or more molecules of interest areionized, and the ions are subsequently introduced into a massspectrographic instrument where, due to a combination of magnetic andelectric fields, the ions follow a path in space that is dependent uponmass (“m”) and charge (“z”). See, e.g., U.S. Pat. No. 6,204,500,entitled “Mass Spectrometry From Surfaces;” U.S. Pat. No. 6,107,623,entitled “Methods and Apparatus for Tandem Mass Spectrometry;” U.S. Pat.No. 6,268,144, entitled “DNA Diagnostics Based On Mass Spectrometry;”U.S. Pat. No. 6,124,137, entitled “Surface-Enhanced PhotolabileAttachment And Release For Desorption And Detection Of Analytes;” Wrightet al., Prostate Cancer and Prostatic Diseases 2:264-76 (1999); andMerchant and Weinberger, Electrophoresis 21:1164-67 (2000).

As used herein, the term “operating in negative ion mode” refers tothose mass spectrometry methods where negative ions are generated anddetected. The term “operating in positive ion mode” as used herein,refers to those mass spectrometry methods where positive ions aregenerated and detected.

As used herein, the term “ionization” or “ionizing” refers to theprocess of generating an analyte ion having a net electrical chargeequal to one or more electron units. Negative ions are those having anet negative charge of one or more electron units, while positive ionsare those having a net positive charge of one or more electron units.

As used herein, the term “electron ionization” or “EI” refers to methodsin which an analyte of interest in a gaseous or vapor phase interactswith a flow of electrons. Impact of the electrons with the analyteproduces analyte ions, which may then be subjected to a massspectrometry technique.

As used herein, the term “chemical ionization” or “CI” refers to methodsin which a reagent gas (e.g.ammonia) is subjected to electron impact,and analyte ions are formed by the interaction of reagent gas ions andanalyte molecules.

As used herein, the term “fast atom bombardment” or “FAB” refers tomethods in which a beam of high energy atoms (often Xe or Ar) impacts anon-volatile sample, desorbing and ionizing molecules contained in thesample. Test samples are dissolved in a viscous liquid matrix such asglycerol, thioglycerol, m-nitrobenzyl alcohol, 18-crown-6 crown ether,2-nitrophenyloctyl ether, sulfolane, diethanolamine, and triethanolamineThe choice of an appropriate matrix for a compound or sample is anempirical process.

As used herein, the term “matrix-assisted laser desorption ionization”or “MALDI” refers to methods in which a non-volatile sample is exposedto laser irradiation, which desorbs and ionizes analytes in the sampleby various ionization pathways, including photo-ionization, protonation,deprotonation, and cluster decay. For MALDI, the sample is mixed with anenergy-absorbing matrix, which facilitates desorption of analytemolecules.

As used herein, the term “surface enhanced laser desorption ionization”or “SELDI” refers to another method in which a non-volatile sample isexposed to laser irradiation, which desorbs and ionizes analytes in thesample by various ionization pathways, including photo-ionization,protonation, deprotonation, and cluster decay. For SELDI, the sample istypically bound to a surface that preferentially retains one or moreanalytes of interest. As in MALDI, this process may also employ anenergy-absorbing material to facilitate ionization.

As used herein, the term “electrospray ionization” or “ESI,” refers tomethods in which a solution is passed along a short length of capillarytube, to the end of which is applied a high positive or negativeelectric potential. Solution reaching the end of the tube is vaporized(nebulized) into a jet or spray of very small droplets of solution insolvent vapor. This mist of droplets flows through an evaporationchamber, which is heated slightly to prevent condensation and toevaporate solvent. As the droplets get smaller the electrical surfacecharge density increases until such time that the natural repulsionbetween like charges causes ions as well as neutral molecules to bereleased.

As used herein, the term “atmospheric pressure chemical ionization” or“APCI,” refers to mass spectroscopy methods that are similar to ESI;however, APCI produces ions by ion-molecule reactions that occur withina plasma at atmospheric pressure. The plasma is maintained by anelectric discharge between the spray capillary and a counter electrode.Then ions are typically extracted into the mass analyzer by use of a setof differentially pumped skimmer stages. A counterflow of dry andpreheated N₂ gas may be used to improve removal of solvent. Thegas-phase ionization in APCI can be more effective than ESI foranalyzing less-polar species.

The term “Atmospheric Pressure Photoionization” or “APPI” as used hereinrefers to the form of mass spectroscopy where the mechanism for thephotoionization of molecule M is photon absorption and electron ejectionto form the molecular ion M+. Because the photon energy typically isjust above the ionization potential, the molecular ion is lesssusceptible to dissociation. In many cases it may be possible to analyzesamples without the need for chromatography, thus saving significanttime and expense. In the presence of water vapor or protic solvents, themolecular ion can extract H to form MH+. This tends to occur if M has ahigh proton affinity. This does not affect quantitation accuracy becausethe sum of M+ and MH+ is constant. Drug compounds in protic solvents areusually observed as MH+, whereas nonpolar compounds such as naphthaleneor testosterone usually form M+. Robb, D. B., Covey, T. R. and Bruins,A. P. (2000): See, e.g., Robb et al., Atmospheric pressurephotoionization: An ionization method for liquid chromatography-massspectrometry. Anal. Chem. 72(15): 3653-3659.

As used herein, the term “inductively coupled plasma” or “ICP” refers tomethods in which a sample interacts with a partially ionized gas at asufficiently high temperature such that most elements are atomized andionized.

As used herein, the term “field desorption” refers to methods in which anon-volatile test sample is placed on an ionization surface, and anintense electric field is used to generate analyte ions.

As used herein, the term “desorption” refers to the removal of ananalyte from a surface and/or the entry of an analyte into a gaseousphase.

As used herein, the term “limit of quantification”, “limit ofquantitation” or “LOQ” refers to the point where measurements becomequantitatively meaningful. The analyte response at this LOQ isidentifiable, discrete and reproducible with a precision of 20% and anaccuracy of 80% to 120%.

As used herein, the term “limit of detection” or “LOD” is the point atwhich the measured value is larger than the uncertainty associated withit. The LOD is defined arbitrarily as 2 standard deviations (SD) fromthe zero concentration.

As used herein, an “amount” of amyloid beta in a body fluid samplerefers generally to an absolute value reflecting the mass of amyloidbeta detectable in volume of body fluid. However, an amount alsocontemplates a relative amount in comparison to another amyloid betaamount. For example, an amount of amyloid beta in a body fluid can be anamount which is greater than or less than a control or normal level ofamyloid beta normally present.

The term “about” as used herein in reference to quantitativemeasurements not including the measurement of the mass of an ion, refersto the indicated value plus or minus 10%. Mass spectrometry instrumentscan vary slightly in determining the mass of a given analyte. The term“about” in the context of the mass of an ion or the mass/charge ratio ofan ion refers to +/−0.5 atomic mass unit.

The summary of the invention described above is non-limiting and otherfeatures and advantages of the invention will be apparent from thefollowing detailed description of the invention, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the sequences of Aβ40 (SEQ ID NO:1), Aβ40 fragment (SEQ IDNO:2), Aβ42 (SEQ ID NO:3), and Aβ42 fragment (SEQ ID NO:4).

FIG. 2 shows the liquid chromatography method information. Mobile phaseA of 0.1% FA in water and mobile phase B of 0.1% FA in ACN were used.

FIG. 3 shows the heated electrospray ionization (HESI) ion sourceconditions.

FIG. 4 shows the linear range of Aβ42 analysis.

FIG. 5 shows the linear range of Aβ40 analysis.

FIGS. 6 and 7 show example patient chromatograms of Aβ40, Aβ42, andinternal standard.

FIG. 8 shows the ratio of Aβ42:Aβ40 in 211 subjects comprising 91Alzheimer's patients, 66 borderline Alzheimer's patients, and normalsubjects.

FIG. 9 shows the ratio of Aβ42:Aβ40 in female vs. male Alzheimer'spatients.

FIG. 10 shows the ratio of Aβ42:Aβ40 in age stratified groups ofAlzheimer's patients.

FIG. 11 shows the sensitivity comparison between C8 versus C4 analyticalcolumns

FIG. 12 shows recovery of Aβ40 from treated versus nontreated tubes.

FIG. 13 shows recovery of Aβ42 from treated versus nontreated tubes.

FIG. 14 shows patient Aβ values (μg/mL) sorted by decreasing Aβ40levels.

FIG. 15 shows patient Aβ values (μg/mL) sorted by decreasing Aβ42levels.

FIG. 16 shows patient Alzheimer's Disease diagnosis sorted by Aβ42/Aβ40values.

FIG. 17 shows patient Alzheimer's Disease diagnosis based on Aβ42/Aβ40ratio.

DETAILED DESCRIPTION OF THE INVENTION

Provided herein are methods for detecting or determining the amount ofamyloid beta (Aβ) in a sample by mass spectrometry, including tandemmass spectrometry. In certain embodiments, the methods provided hereinfor determining the amount of amyloid beta comprises (a) purifyingamyloid beta in the sample; (b) ionizing amyloid beta in the sample; and(c) determining the amount of the amyloid beta ion(s) by massspectrometry; wherein the amount of the amyloid beta ion(s) is relatedto the amount of amyloid beta in the sample.

In certain embodiments, the methods provided herein for determining theamount of amyloid beta comprises (a) purifying amyloid beta in thesample; (b) ionizing amyloid beta in the sample to produce a precursorion of amyloid beta; (c) generating one or more fragment ions of amyloidbeta; and (d) determining the amount of the ion(s) from step (c) or (d)or both by mass spectrometry; wherein the amount of the amyloid betaion(s) is related to the amount of amyloid beta in the sample.

In certain embodiments, the methods provided herein for determining theamount of amyloid beta comprises (a) digesting amyloid beta in thesample to generate one or more fragments of amyloid beta; (b) purifyingthe one or more amyloid beta fragments; (c) ionizing amyloid beta in thesample to produce a precursor ion of amyloid beta; (d) generating one ormore fragment ions of amyloid beta; and (e) determining the amount ofthe ion(s) from step (c) or (d) or both by mass spectrometry; whereinthe amount of the amyloid beta ion(s) is related to the amount ofamyloid beta in the sample.

In certain embodiments, provided herein are methods for determining theamount of amyloid beta 42 (Aβ42). In some embodiments, the Aβ42 fragmentcomprises the sequence GAIIGLMVGGVVIA (SEQ ID NO:4). In someembodiments, the methods comprise (a) digesting amyloid beta in thesample to generate amyloid beta 42 (Aβ42); (b) purifying Aβ42; (c)ionizing Aβ42 to produce a precursor ion; (d) generating one or morefragment ions of Aβ42; and (e) determining the amount of the ion(s) fromstep (c) or (d) or both by mass spectrometry; wherein the amount of theion(s) is related to the amount of Aβ42 in the sample.

In certain embodiments, provided herein are methods for determining theamount of amyloid beta 40 (Aβ40). In some embodiments, the Aβ40 fragmentcomprises the sequence GAIIGLMVGGVV (SEQ ID NO:2). In some embodiments,the methods comprise (a) digesting amyloid beta in the sample togenerate amyloid beta 40 (Aβ40); (b) purifying Aβ40; (c) ionizing Aβ40to produce a precursor ion; (d) generating one or more fragment ions ofAβ40; and (e) determining the amount of the ion(s) from step (c) or (d)or both by mass spectrometry; wherein the amount of the ion(s) isrelated to the amount of Aβ40 in the sample.

In certain embodiments, provided herein are methods for determining theamount of amyloid beta 42 (Aβ42) and amyloid beta 40 (Aβ40). In someembodiments, the methods comprise (a) digesting amyloid beta in thesample to generate amyloid beta 42 (Aβ42) and amyloid beta 40 (Aβ40);(b) purifying Aβ42 and Aβ40; (c) ionizing Aβ42 and Aβ40 to produceprecursor ions; (d) generating one or more fragment ions of Aβ42 andAβ40; and (e) determining the amount of the ion(s) from step (c) or (d)or both by mass spectrometry; wherein the amount of the ion(s) isrelated to the amount of Aβ42 and Aβ40 in the sample.

In certain embodiments, provided herein are methods for determining theratio of amyloid beta 42 (Aβ42) to amyloid beta 40 (Aβ40). In someembodiments, the methods comprise (a) digesting amyloid beta in thesample to generate amyloid beta 42 (Aβ42) and amyloid beta 40 (Aβ40);(b) purifying Aβ42 and Aβ40; (c) ionizing Aβ42 and Aβ40 to produceprecursor ions; (d) generating one or more fragment ions of Aβ42 andAβ40; and (e) determining the amount of the ion(s) from step (c) or (d)or both by mass spectrometry; and (f) determining the ratio of Aβ42 toAβ40. In some embodiments, the methods comprise determining the ratio ofAβ40 to Aβ42.

In certain embodiments, provided herein are methods for diagnosis orprognosis of Alzheimer's disease or dementia, the method comprisingdetermining the amount of amyloid beta in a test sample by massspectrometry; wherein an abnormal levels of amyloid beta is predictiveor diagnostic of Alzheimer's disease. In some embodiments, the methodsmay include: (a) purifying amyloid beta in the sample; (b) ionizingamyloid beta in the sample; and (c) determining the amount of theamyloid beta ion(s) by mass spectrometry; and (d) the amount of theamyloid beta ion(s) is related to the amount of amyloid beta in thesample; wherein the abnormal levels of amyloid beta is predictive ordiagnostic of Alzheimer's disease. In some embodiments, the methodscomprise determining the ratio of amyloid beta fragments. In someembodiments, the methods comprise determining the ratio of amyloid beta42 (Aβ42) to amyloid beta 40 (Aβ40). In some embodiments, the methodscomprise determining the ratio of amyloid beta 42 (Aβ40) to amyloid beta40 (Aβ42).

In certain embodiments, the methods provided herein comprise pretreatingsurfaces of equipment that come in contact with the sample. In someembodiments, the pretreatment comprises pre-coating the surfaces ofequipment with an agent that prevents amyloid beta or fragments thereoffrom sticking to the surfaces. In some embodiments, the pretreatmentcomprises bacterial lysate pretreatment. In some embodiments, thepretreatment comprises E. coli lysate pretreatment. In some embodiments,the E. coli lysate comprises a trypsin-digested E. coli lysate. In someembodiments, the pretreated equipment includes, but not limited to, testtubes or plates, pipette tips, sample preparation apparatus, liquidchromatography apparatus, and mass spectrometry apparatus.

In certain embodiments, the methods provided herein comprise treating orincubating the sample with an agent that stabilizes amyloid beta orfragments thereof. In some embodiments, the methods provided hereincomprise treating or incubating the sample with an amyloid betaantibody. In some embodiments, the methods provided herein comprisetreating or incubating the sample with at least two distinct amyloidbeta antibodies. In some embodiments, the amyloid beta antibodycomprises an antibody that binds to the C-terminus of amyloid beta. Insome embodiments, the amyloid beta antibody comprises an antibody thatbinds to the N-terminus of amyloid beta. In some embodiments, the agentthat stabilizes amyloid beta comprises an apolipoprotein. In someembodiments, the agent that stabilizes amyloid beta comprisesapolipoprotein E2. In some embodiments, the agent that stabilizesamyloid beta comprises apolipoprotein E4. In some embodiments, the agentthat stabilizes amyloid beta comprises an antibody that binds to theC-terminus of amyloid beta, an antibody that binds to the N-terminus ofamyloid beta, apolipoprotein E2, apolipoprotein E4, or a combinationthereof. In some embodiments, the agent that stabilizes amyloid betaprovided herein confers stability for at least 1 month at −70° C. Insome embodiments, the agent that stabilizes amyloid beta provided hereinconfers stability for at least 2 months at −70° C. In some embodiments,the agent that stabilizes amyloid beta provided herein confers stabilityfor at least 3 months at −70° C. In some embodiments, the agent thatstabilizes amyloid beta provided herein confers stability through afreeze-thaw cycle. In some embodiments, the agent that stabilizesamyloid beta provided herein confers stability through at least twofreeze-thaw cycles. In some embodiments, the agent that stabilizesamyloid beta provided herein confers stability through at least threefreeze-thaw cycles. In some embodiments, the agent that stabilizesamyloid beta provided herein confers stability through at least fourfreeze-thaw cycles.

In certain embodiments, the methods provided herein comprise digestingamyloid beta in the sample. In some embodiments, the methods providedherein comprise digesting amyloid beta with an enzyme. In someembodiments, the enzyme is Lys-C. In some embodiments, the methodsprovided herein comprise digesting amyloid beta with urea. In someembodiments, the urea is in a concentration suitable for proteindigestion. In some embodiments, the urea is 6M urea. In someembodiments, the methods provided herein comprise digesting amyloid betawith urea and Lys-C. In some embodiments, the digestion comprisesdigesting in conditions that reduce digestion time or increase digestionefficiency. In some embodiments, the digestion comprises digesting inmicrowave.

In certain embodiments, the methods provided herein comprise anextraction. In some embodiments, the methods provided herein comprise amixed mode anion exchange extraction. In some embodiments, the methodsprovided herein comprise a solid phase extraction.

In certain embodiments, the methods provided herein comprise eluting anddrying the sample using heated nitrogen. In some embodiments, the sampleis resuspended in a reconstitution buffer.

In certain embodiments, the purifying the sample comprises a liquidchromatography. In some embodiments, liquid chromatography includes, butnot limited to, reverse phase liquid chromatography (RPLC), highperformance liquid chromatography (HPLC), and high turbulence liquidchromatography (HTLC). In a preferred embodiment, liquid chromatographycomprises HPLC. In some embodiments, HPLC column typically includes amedium (i.e., a packing material) to facilitate separation of chemicalmoieties (i.e., fractionation). Suitable columns may include C-4, C-8,C-12, or C-18 columns. In a preferred embodiment, a suitable HPLC columnis C-4 column.

In certain embodiments, the methods provided herein comprise usingequipment that reduces sticking of amyloid beta to the surfaces ofequipment. In some embodiments, the equipment comprises PEEK (poly etherether ketone) tubing or apparatus. In some embodiments, the equipmentcomprises metal tubing or apparatus.

In certain embodiments, the methods provided herein comprise tandem massspectrometry. In some embodiments, the methods provided herein compriseionizing in positive mode. In some embodiments, the methods providedherein comprise ionizing in negative mode. In some embodiments, themethods provided herein comprise ionizing using heated electrosprayionization (HESI). In some embodiments, the methods provided hereincomprise ionizing using electrospray ionization (ESI). In someembodiments, the methods provided herein comprise ionizing usingatmospheric pressure chemical ionization (APCI). In a preferredembodiment, the methods provided herein comprise ionizing using heatedelectrospray ionization (HESI) in positive mode. In some embodiments,the collision energy is between 5V to 60V. In some embodiments, thecollision energy is between 10V to 50V. In some embodiments, thecollision energy is between 20V to 50V. In some embodiments, thecollision energy is between 20V to 45V.

In certain embodiments, the methods provided herein comprise detectingor determining the amount of amyloid beta 40 (Aβ40). In someembodiments, Aβ40 comprises the sequenceDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV (SEQ ID NO:1). In someembodiments, the methods provided herein comprise detecting ordetermining the amount of a fragment of Aβ40. In some embodiments, theAβ40 fragment comprises the sequence GAIIGLMVGGVV (SEQ ID NO:2). In someembodiments, the Aβ40 fragment comprises a sequence containing anN-terminal or C-terminal winged peptide. In some embodiments, the Aβ40fragment comprises SEQ ID NO:2 and an N-terminal or C-terminal wingedpeptide. In some embodiments, the winged peptide is hydrophilic. In someembodiments, the winged peptide comprises at least one amino acid. Insome embodiments, the winged peptide comprises at least two amino acids.In some embodiments, the winged peptide comprises at least three aminoacids. In some embodiments, the winged peptide comprises at least fouramino acids. In some embodiments, the winged peptide comprises at leastfive amino acids. In some embodiments, the winged peptide comprises atleast six amino acids. In some embodiments, the amount of the Aβ40fragment correlates to the amount of Aβ40 in the sample.

In certain embodiments, the methods provided herein comprise detectingor determining the amount of amyloid beta 42 (Aβ42). In someembodiments, Aβ42 comprises the sequenceDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA (SEQ ID NO:3). In someembodiments, the methods provided herein comprise detecting ordetermining the amount of a fragment of Aβ42. In some embodiments, theAβ42 fragment comprises the sequence GAIIGLMVGGVVIA (SEQ ID NO:4). Insome embodiments, the Aβ42 fragment comprises a sequence containing anN-terminal or C-terminal winged peptide. In some embodiments, the Aβ42fragment comprises SEQ ID NO:4 and an N-terminal or C-terminal wingedpeptide. In some embodiments, the winged peptide is hydrophilic. In someembodiments, the winged peptide comprises at least one amino acid. Insome embodiments, the winged peptide comprises at least two amino acids.In some embodiments, the winged peptide comprises at least three aminoacids. In some embodiments, the winged peptide comprises at least fouramino acids. In some embodiments, the winged peptide comprises at leastfive amino acids. In some embodiments, the winged peptide comprises atleast six amino acids. In some embodiments, the amount of the Aβ42fragment correlates to the amount of Aβ42 in the sample.

In certain embodiments, the methods provided herein comprise detectingor determining the ratio of Aβ40 to Aβ42 (Aβ40:Aβ42). In someembodiments, the methods provided herein comprise detecting ordetermining the ratio of the Aβ40 fragment to the Aβ42 fragment (Aβ40fragment:Aβ42 fragment). In some embodiments, the methods providedherein comprise detecting or determining the ratio of Aβ42 to Aβ40(Aβ42:Aβ40). In some embodiments, the methods provided herein comprisedetecting or determining the ratio of the Aβ42 fragment to the Aβ40fragment (Aβ42 fragment:Aβ40 fragment).

In certain embodiments, the ratio of Aβ42 to Aβ40, or the ratio of theAβ42 fragment to the Aβ40 fragment, of 0.6 or less is predictive ordiagnostic of Alzheimer's disease. In some embodiments, the ratio ofAβ42 to Aβ40, or the ratio of the Aβ42 fragment to the Aβ40 fragment, of0.5 or less is predictive or diagnostic of Alzheimer's disease. In someembodiments, the ratio of Aβ42 to Aβ40, or the ratio of the Aβ42fragment to the Aβ40 fragment, of 0.45 or less is predictive ordiagnostic of Alzheimer's disease. In some embodiments, the ratio ofAβ42 to Aβ40, or the ratio of the Aβ42 fragment to the Aβ40 fragment, of0.4 or less is predictive or diagnostic of Alzheimer's disease. In someembodiments, the ratio of Aβ42 to Aβ40, or the ratio of the Aβ42fragment to the Aβ40 fragment, of 0.35 or less is predictive ordiagnostic of Alzheimer's disease. In some embodiments, the ratio ofAβ42 to Aβ40, or the ratio of the Aβ42 fragment to the Aβ40 fragment, of0.3 or less is predictive or diagnostic of Alzheimer's disease. In someembodiments, the ratio of Aβ42 to Aβ40, or the ratio of the Aβ42fragment to the Aβ40 fragment, of 0.25 or less is predictive ordiagnostic of Alzheimer's disease. In some embodiments, the ratio ofAβ42 to Aβ40, or the ratio of the Aβ42 fragment to the Aβ40 fragment, of0.2 or less is predictive or diagnostic of Alzheimer's disease. In someembodiments, the ratio of Aβ42 to Aβ40, or the ratio of the Aβ42fragment to the Aβ40 fragment, of 0.19 or less is predictive ordiagnostic of Alzheimer's disease. In some embodiments, the ratio ofAβ42 to Aβ40, or the ratio of the Aβ42 fragment to the Aβ40 fragment, of0.18 or less is predictive or diagnostic of Alzheimer's disease. In someembodiments, the ratio of Aβ42 to Aβ40, or the ratio of the Aβ42fragment to the Aβ40 fragment, of 0.17 or less is predictive ordiagnostic of Alzheimer's disease. In some embodiments, the ratio ofAβ42 to Aβ40, or the ratio of the Aβ42 fragment to the Aβ40 fragment, of0.16 or less is predictive or diagnostic of Alzheimer's disease. In someembodiments, the ratio of Aβ42 to Aβ40, or the ratio of the Aβ42fragment to the Aβ40 fragment, of 0.15 or less is predictive ordiagnostic of Alzheimer's disease. In some embodiments, the ratio ofAβ42 to Aβ40, or the ratio of the Aβ42 fragment to the Aβ40 fragment, of0.1 or less is predictive or diagnostic of Alzheimer's disease.

In certain embodiments, the methods include generating one or moreprecursor ions of AP or a fragment thereof. In some embodiments, atleast one of the precursor ions has a mass/charge ratio of 1085.6±0.5 or1269.7±0.5. In some embodiments, the methods may include generating oneor more fragment ions of Aβ or a fragment thereof. In some embodiments,at least one of the fragment ions has a mass/charge ratio of 812.37±0.5,869.4±0.5, 968.43±0.5, 869.39±0.5, 968.44±0.5, 1067.5±0.5, or1180.57±0.5.

In certain embodiments, the methods provided herein comprise adding aninternal standard. In some embodiments, the internal standard comprisesan isotopically labeled internal standard. In some embodiments, theinternal standard comprises ¹³C¹⁵N labeling. In some embodiments, theinternal standard comprises at least one Phe, Leu, or Met labeled with¹³C¹⁵N. In some embodiments, at least one of the precursor ions of theinternal standard has a mass/charge ratio of 1110.7±0.5. In someembodiments, the methods may include generating one or more fragmentions of the internal standard. In some embodiments, at least one of thefragment ions has a mass/charge ratio of 768.48±0.5, 825.5±0.5, or882.52±0.5.

In certain embodiments, the limit of quantitation of the methods is lessthan or equal to 10 ng/mL. In some embodiments, the limit ofquantitation of the methods is less than or equal to 5 ng/mL. In someembodiments, the limit of quantitation of the methods is less than orequal to 4 ng/mL. In some embodiments, the limit of quantitation of themethods is less than or equal to 3 ng/mL. In some embodiments, the limitof quantitation of the methods is less than or equal to 2 ng/mL. In someembodiments, the limit of quantitation of the methods is less than orequal to 1 ng/mL. In some embodiments, the limit of quantitation of themethods is less than or equal to 0.5 ng/mL. In some embodiments, thelimit of quantitation of the methods is less than or equal to 0.2 ng/mL.In some embodiments, the limit of quantitation of the methods is lessthan or equal to 0.1 ng/mL.

In some embodiments, the limit of detection of the methods is less thanor equal to 5 ng/mL. In some embodiments, the limit of detection of themethods is less than or equal to 1 ng/mL. In some embodiments, the limitof detection of the methods is less than or equal to 0.5 ng/mL. In someembodiments, the limit of detection of the methods is less than or equalto 0.1 ng/mL. In some embodiments, the limit of detection of the methodsis less than or equal to 0.05 ng/mL. In some embodiments, the limit ofdetection of the methods is less than or equal to 0.01 ng/mL.

In some embodiments, amyloid beta is not derivatized prior to massspectrometry. In some embodiments, amyloid beta is derivatized prior tomass spectrometry.

In certain embodiments, the sample is a body fluid. In some embodiments,the sample is cerebrospinal fluid (CSF). In some embodiments, the sampleis plasma or serum. In some embodiments, the sample is whole blood. Insome embodiments, the sample is saliva or urine.

Suitable test samples include any test sample that may contain theanalyte of interest. In some preferred embodiments, a sample is abiological sample; that is, a sample obtained from any biologicalsource, such as an animal, a cell culture, an organ culture, etc. Incertain preferred embodiments samples are obtained from a mammaliananimal, such as a dog, cat, horse, etc. Particularly preferred mammaliananimals are primates, most preferably male or female humans.Particularly preferred samples include blood, plasma, serum, hair,muscle, urine, saliva, tear, cerebrospinal fluid, or other tissuesample. Such samples may be obtained, for example, from a patient; thatis, a living person, male or female, presenting oneself in a clinicalsetting for diagnosis, prognosis, or treatment of a disease orcondition. The test sample is preferably obtained from a patient, forexample, blood serum.

Sample Preparation for Mass Spectrometry

Methods that may be used to enrich in amyloid beta relative to othercomponents in the sample (e.g. protein) include for example, filtration,centrifugation, thin layer chromatography (TLC), electrophoresisincluding capillary electrophoresis, affinity separations includingimmunoaffinity separations, extraction methods including ethyl acetateextraction and methanol extraction, and the use of chaotropic agents orany combination of the above or the like.

Protein precipitation is one preferred method of preparing a testsample. Such protein purification methods are well known in the art, forexample, Polson et al., Journal of Chromatography B 785:263-275 (2003),describes protein precipitation techniques suitable for use in themethods. Protein precipitation may be used to remove most of the proteinfrom the sample leaving amyloid beta in the supernatant. The samples maybe centrifuged to separate the liquid supernatant from the precipitatedproteins. The resultant supernatant may then be applied to liquidchromatography and subsequent mass spectrometry analysis. In certainembodiments, the use of protein precipitation such as for example,acetonitrile protein precipitation, obviates the need for highturbulence liquid chromatography (HTLC) or other on-line extractionprior to HPLC and mass spectrometry. Accordingly in such embodiments,the method involves (1) performing a protein precipitation of the sampleof interest; and (2) loading the supernatant directly onto the HPLC-massspectrometer without using on-line extraction or high turbulence liquidchromatography (HTLC).

In some preferred embodiments, HPLC, alone or in combination with one ormore purification methods, may be used to purify amyloid beta prior tomass spectrometry. In such embodiments samples may be extracted using anHPLC extraction cartridge which captures the analyte, then eluted andchromatographed on a second HPLC column or onto an analytical HPLCcolumn prior to ionization. Because the steps involved in thesechromatography procedures can be linked in an automated fashion, therequirement for operator involvement during the purification of theanalyte can be minimized This feature can result in savings of time andcosts, and eliminate the opportunity for operator error.

It is believed that turbulent flow, such as that provided by HTLCcolumns and methods, may enhance the rate of mass transfer, improvingseparation characteristics. HTLC columns separate components by means ofhigh chromatographic flow rates through a packed column containing rigidparticles. By employing high flow rates (e.g., 3-5 mL/min), turbulentflow occurs in the column that causes nearly complete interactionbetween the stationary phase and the analyte(s) of interest. Anadvantage of using HTLC columns is that the macromolecular build-upassociated with biological fluid matrices is avoided since the highmolecular weight species are not retained under the turbulent flowconditions. HTLC methods that combine multiple separations in oneprocedure lessen the need for lengthy sample preparation and operate ata significantly greater speed. Such methods also achieve a separationperformance superior to laminar flow (HPLC) chromatography. HTLC allowsfor direct injection of biological samples (plasma, urine, etc.). Directinjection is difficult to achieve in traditional forms of chromatographybecause denatured proteins and other biological debris quickly block theseparation columns. HTLC also allows for very low sample volume of lessthan 1 mL, preferably less than 0.5 mL, preferably less than 0.2 mL,preferably 0.1 mL.

Examples of HTLC applied to sample preparation prior to analysis by massspectrometry have been described elsewhere. See, e.g., Zimmer et al., J.Chromatogr. A 854:23-35 (1999); see also, U.S. Pat. Nos. 5,968,367;5,919,368; 5,795,469; and 5,772,874. In certain embodiments of themethod, samples are subjected to protein precipitation as describedabove prior to loading on the HTLC column; in alternative preferredembodiments, the samples may be loaded directly onto the HTLC withoutbeing subjected to protein precipitation. The HTLC extraction column ispreferably a large particle column. In various embodiments, one of moresteps of the methods may be performed in an on-line, automated fashion.For example, in one embodiment, steps (i)-(v) are performed in anon-line, automated fashion. In another, the steps of ionization anddetection are performed on-line following steps (i)-(v).

Liquid chromatography (LC) including high-performance liquidchromatography (HPLC) relies on relatively slow, laminar flowtechnology. Traditional HPLC analysis relies on column packings in whichlaminar flow of the sample through the column is the basis forseparation of the analyte of interest from the sample. The skilledartisan will understand that separation in such columns is a diffusionalprocess. HPLC has been successfully applied to the separation ofcompounds in biological samples but a significant amount of samplepreparation is required prior to the separation and subsequent analysiswith a mass spectrometer (MS), making this technique labor intensive. Inaddition, most HPLC systems do not utilize the mass spectrometer to itsfullest potential, allowing only one HPLC system to be connected to asingle MS instrument, resulting in lengthy time requirements forperforming a large number of assays.

Various methods have been described for using HPLC for sample clean-upprior to mass spectrometry analysis. See, e.g., Taylor et al.,Therapeutic Drug Monitoring 22:608-12 (2000); and Salm et al., Clin.Therapeutics 22 Supl. B:B71-B85 (2000).

One of skill in the art may select HPLC instruments and columns that aresuitable for use with amyloid beta. The chromatographic column typicallyincludes a medium (i.e., a packing material) to facilitate separation ofchemical moieties (i.e., fractionation). The medium may include minuteparticles. The particles include a bonded surface that interacts withthe various chemical moieties to facilitate separation of the chemicalmoieties. One suitable bonded surface is a hydrophobic bonded surfacesuch as an alkyl bonded surface. Alkyl bonded surfaces may include C-4,C-8, C-12, or C-18 bonded alkyl groups, preferably C-18 bonded groups.The chromatographic column includes an inlet port for receiving a sampleand an outlet port for discharging an effluent that includes thefractionated sample. In one embodiment, the sample (or pre-purifiedsample) is applied to the column at the inlet port, eluted with asolvent or solvent mixture, and discharged at the outlet port. Differentsolvent modes may be selected for eluting the analyte(s) of interest.For example, liquid chromatography may be performed using a gradientmode, an isocratic mode, or a polytyptic (i.e. mixed) mode. Duringchromatography, the separation of materials is effected by variablessuch as choice of eluent (also known as a “mobile phase”), elution mode,gradient conditions, temperature, etc.

In certain embodiments, an analyte may be purified by applying a sampleto a column under conditions where the analyte of interest is reversiblyretained by the column packing material, while one or more othermaterials are not retained. In these embodiments, a first mobile phasecondition can be employed where the analyte of interest is retained bythe column, and a second mobile phase condition can subsequently beemployed to remove retained material from the column, once thenon-retained materials are washed through. Alternatively, an analyte maybe purified by applying a sample to a column under mobile phaseconditions where the analyte of interest elutes at a differential ratein comparison to one or more other materials. Such procedures may enrichthe amount of one or more analytes of interest relative to one or moreother components of the sample.

In one preferred embodiment, the HTLC may be followed by HPLC on ahydrophobic column chromatographic system. In certain preferredembodiments, a TurboFlow Cyclone P® polymer-based column from CohesiveTechnologies (60 μm particle size, 50×1.0 mm column dimensions, 100 Åpore size) is used. In related preferred embodiments, a SynergiPolar-RP® ether-linked phenyl, analytical column from Phenomenex Inc (4μm particle size, 150×2.0 mm column dimensions, 80 Å pore size) withhydrophilic endcapping is used. In certain preferred embodiments, HTLCand HPLC are performed using HPLC Grade Ultra Pure Water and 100%methanol as the mobile phases.

By careful selection of valves and connector plumbing, two or morechromatography columns may be connected as needed such that material ispassed from one to the next without the need for any manual steps. Inpreferred embodiments, the selection of valves and plumbing iscontrolled by a computer pre-programmed to perform the necessary steps.Most preferably, the chromatography system is also connected in such anon-line fashion to the detector system, e.g., an MS system. Thus, anoperator may place a tray of samples in an autosampler, and theremaining operations are performed under computer control, resulting inpurification and analysis of all samples selected.

In certain preferred embodiments, amyloid beta or fragments thereof in asample may be purified prior to ionization. In particularly preferredembodiments the chromatography is not gas chromatography.

Detection and Quantitation by Mass Spectrometry

In various embodiments, amyloid beta or fragments thereof may be ionizedby any method known to the skilled artisan. Mass spectrometry isperformed using a mass spectrometer, which includes an ion source forionizing the fractionated sample and creating charged molecules forfurther analysis. For example ionization of the sample may be performedby electron ionization, chemical ionization, electrospray ionization(ESI), photon ionization, atmospheric pressure chemical ionization(APCI), photoionization, atmospheric pressure photoionization (APPI),fast atom bombardment (FAB), liquid secondary ionization (LSI), matrixassisted laser desorption ionization (MALDI), field ionization, fielddesorption, thermospray/plasmaspray ionization, surface enhanced laserdesorption ionization (SELDI), inductively coupled plasma (ICP) andparticle beam ionization. The skilled artisan will understand that thechoice of ionization method may be determined based on the analyte to bemeasured, type of sample, the type of detector, the choice of positiveversus negative mode, etc.

In preferred embodiments, amyloid beta or a fragment thereof is ionizedby heated electrospray ionization (HESI) in positive or negative mode.In alternative embodiments, amyloid beta or a fragment thereof isionized by electrospray ionization (ESI) or atmospheric pressurechemical ionization (APCI) in positive or negative mode.

After the sample has been ionized, the positively charged or negativelycharged ions thereby created may be analyzed to determine amass-to-charge ratio. Suitable analyzers for determining mass-to-chargeratios include quadrupole analyzers, ion traps analyzers, andtime-of-flight analyzers. The ions may be detected using severaldetection modes. For example, selected ions may be detected i.e., usinga selective ion monitoring mode (SIM), or alternatively, ions may bedetected using a scanning mode, e.g., multiple reaction monitoring (MRM)or selected reaction monitoring (SRM). Preferably, the mass-to-chargeratio is determined using a quadrupole analyzer. For example, in a“quadrupole” or “quadrupole ion trap” instrument, ions in an oscillatingradio frequency field experience a force proportional to the DCpotential applied between electrodes, the amplitude of the RF signal,and the mass/charge ratio. The voltage and amplitude may be selected sothat only ions having a particular mass/charge ratio travel the lengthof the quadrupole, while all other ions are deflected. Thus, quadrupoleinstruments may act as both a “mass filter” and as a “mass detector” forthe ions injected into the instrument.

One may enhance the resolution of the MS technique by employing “tandemmass spectrometry,” or “MS/MS”. In this technique, a precursor ion (alsocalled a parent ion) generated from a molecule of interest can befiltered in an MS instrument, and the precursor ion is subsequentlyfragmented to yield one or more fragment ions (also called daughter ionsor product ions) that are then analyzed in a second MS procedure. Bycareful selection of precursor ions, only ions produced by certainanalytes are passed to the fragmentation chamber, where collisions withatoms of an inert gas produce the fragment ions. Because both theprecursor and fragment ions are produced in a reproducible fashion undera given set of ionization/fragmentation conditions, the MS/MS techniquemay provide an extremely powerful analytical tool. For example, thecombination of filtration/fragmentation may be used to eliminateinterfering substances, and may be particularly useful in complexsamples, such as biological samples.

The mass spectrometer typically provides the user with an ion scan; thatis, the relative abundance of each ion with a particular mass/chargeover a given range (e.g., 100 to 1000 amu). The results of an analyteassay, that is, a mass spectrum, may be related to the amount of theanalyte in the original sample by numerous methods known in the art. Forexample, given that sampling and analysis parameters are carefullycontrolled, the relative abundance of a given ion may be compared to atable that converts that relative abundance to an absolute amount of theoriginal molecule. Alternatively, molecular standards may be run withthe samples, and a standard curve constructed based on ions generatedfrom those standards. Using such a standard curve, the relativeabundance of a given ion may be converted into an absolute amount of theoriginal molecule. In certain preferred embodiments, an internalstandard is used to generate a standard curve for calculating thequantity of amyloid beta. Methods of generating and using such standardcurves are well known in the art and one of ordinary skill is capable ofselecting an appropriate internal standard. For example, an isotope ofamyloid beta may be used as an internal standard. Numerous other methodsfor relating the amount of an ion to the amount of the original moleculewill be well known to those of ordinary skill in the art.

One or more steps of the methods may be performed using automatedmachines. In certain embodiments, one or more purification steps areperformed on-line, and more preferably all of the purification and massspectrometry steps may be performed in an on-line fashion.

In certain embodiments, such as MS/MS, where precursor ions are isolatedfor further fragmentation, collision activation dissociation is oftenused to generate the fragment ions for further detection. In CAD,precursor ions gain energy through collisions with an inert gas, andsubsequently fragment by a process referred to as “unimoleculardecomposition”. Sufficient energy must be deposited in the precursor ionso that certain bonds within the ion can be broken due to increasedvibrational energy.

In particularly preferred embodiments, amyloid beta is detected and/orquantified using MS/MS as follows. The samples are subjected to liquidchromatography, preferably HPLC, the flow of liquid solvent from thechromatographic column enters the heated nebulizer interface of an MS/MSanalyzer and the solvent/analyte mixture is converted to vapor in theheated tubing of the interface. The analyte is ionized by the selectedionizer. The ions, e.g. precursor ions, pass through the orifice of theinstrument and enter the first quadrupole. Quadrupoles 1 and 3 (Q1 andQ3) are mass filters, allowing selection of ions (i.e., “precursor” and“fragment” ions) based on their mass to charge ratio (m/z). Quadrupole 2(Q2) is the collision cell, where ions are fragmented. The firstquadrupole of the mass spectrometer (Q1) selects for molecules with themass to charge ratios of amyloid beta. Precursor ions with the correctmass/charge ratios of amyloid beta are allowed to pass into thecollision chamber (Q2), while unwanted ions with any other mass/chargeratio collide with the sides of the quadrupole and are eliminated.Precursor ions entering Q2 collide with neutral argon gas molecules andfragment. This process is called collision activated dissociation (CAD).The fragment ions generated are passed into quadrupole 3 (Q3), where thefragment ions of amyloid beta are selected while other ions areeliminated.

The methods may involve MS/MS performed in either positive or negativeion mode. Using standard methods well known in the art, one of ordinaryskill is capable of identifying one or more fragment ions of aparticular precursor ion of amyloid beta that may be used for selectionin quadrupole 3 (Q3).

If the precursor ion of amyloid beta includes an alcohol or amine group,fragment ions are commonly formed that represent dehydration ordeamination of the precursor ion, respectfully. In the case of precursorions that include an alcohol group, such fragment ions formed bydehydration are caused by a loss of one or more water molecules from theprecursor ion (i.e., where the difference in mass to charge ratiobetween the precursor ion and fragment ion is about 18 for the loss ofone water molecule, or about 36 for the loss of two water molecules,etc.). In the case of precursor ions that include an amine group, suchfragment ions formed by deamination are caused by a loss of one or moreammonia molecules (i.e. where the difference in mass to charge ratiobetween the precursor ion and fragment ion is about 17 for the loss ofone ammonia molecule, or about 34 for the loss of two ammonia molecules,etc.). Likewise, precursor ions that include one or more alcohol andamine groups commonly form fragment ions that represent the loss of oneor more water molecules and/or one or more ammonia molecules (i.e.,where the difference in mass to charge ratio between the precursor ionand fragment ion is about 35 for the loss of one water molecule and theloss of one ammonia molecule). Generally, the fragment ions thatrepresent dehydrations or deaminations of the precursor ion are notspecific fragment ions for a particular analyte. Accordingly, inpreferred embodiments of the invention, MS/MS is performed such that atleast one fragment ion of amyloid beta is detected that does notrepresent only a loss of one or more water molecules and/or a loss ofone or more ammonia molecules from the precursor ion.

As ions collide with the detector they produce a pulse of electrons thatare converted to a digital signal. The acquired data is relayed to acomputer, which plots counts of the ions collected versus time. Theresulting mass chromatograms are similar to chromatograms generated intraditional HPLC methods. The areas under the peaks corresponding toparticular ions, or the amplitude of such peaks, are measured and thearea or amplitude is correlated to the amount of the analyte ofinterest. In certain embodiments, the area under the curves, oramplitude of the peaks, for fragment ion(s) and/or precursor ions aremeasured to determine the amount of amyloid beta. As described above,the relative abundance of a given ion may be converted into an absoluteamount of the original analyte, using calibration standard curves basedon peaks of one or more ions of an internal molecular standard.

The following examples serve to illustrate the invention. These examplesare in no way intended to limit the scope of the methods.

EXAMPLES Example 1: Equipment Pretreatment and Amyloid BetaStabilization 96 Well Polypropylene Plate and Microcentrifuge Test TubePretreatment

E. coli was lysed and precipitated using methanol in a 96 well plate.

Methanol was then discarded while the pellet is resuspended in ammoniumbicarbonate pH 8.

Sigma bovine trypsin was added, and the samples were incubated at 60° C.for 2 days.

All digest was then discarded to waste or pooled to pretreat pipettetips.

Empty plates and test tubes were completely dried.

Pipette Tip Pre-Treatment

Pipette tips were used to pipette up and down E. coli digest at least 3times and then incubated at 60° C. for 2 days or until completely dried.

Amyloid Beta Peptide Stabilization

AB40, AB42, and internal standards were synthesized in pretreated testtubes and were resuspended in 6M urea with 4 mg BSA.

The standards were then individually diluted 1:10 in 0.1M PBS withN-terminal and C-terminal antibodies and apolipoprotein E2 and E4.

Each mixture was incubated at room temp on a shaker for 1 hour.

AB40 and AB42 was then combined and diluted down in 0.1M PBS and 0.4mg/mL BSA to make high calibrator standards before being frozen.

The table below shows a side-by-side comparison of the same sampleprepped in a treated and non-treated vial.

AB42 Treated-Calc. value Non-treated-Calc. value % Recovery AB212 2.520.33 13.10 AB213 2.14 0.81 37.85 AB214 5.73 1.45 25.31 AB215 0.92 0.2122.83 AB216 1.85 0.99 53.51 AB40 Treated-Calc. value Non-treated-Calc.value AB212 15.01 2.73 18.19 AB213 13.11 4.93 37.60 AB214 19.18 5.6229.30 AB215 6.02 2.23 37.04 AB216 7.08 4.76 67.23

Example 2: Sample Preparation

0.5 mL of sample or standard was pipetted using pre-treated pipette tipsinto a pre-treated 96 well plates.

5 ng of internal standard was added.

250 uL of 18M urea was added.

2 ug of Lys-C was added to each sample for digestion of amyloid beta.

Plate was sealed with an adhesive lid before being digested in aenzymatic microwave at 450 w, 4 ° C. for 4 hours.

Samples were then extracted using Waters mixed mode strong anionexchange.

Sample eluates were then dried down completely using heated nitrogen.

Samples were then resuspended in a reconstitution buffer for LC-MS/MSanalysis.

Example 3: Detection and Quantitation of Amyloid Beta Fragments by MS/MS

Ions passed to the first quadrupole (Q1), which selected ions with amass to charge ratio of either 1085.6±0.5 m/z or 1269.7±.5 m/z for theAβ40 fragment and the Aβ42 fragment, respectively. Ions enteringQuadrupole 2 (Q2) collided with argon gas to generate ion fragments,which were passed to quadrupole 3 (Q3) for further selection.Simultaneously, the same process using isotope dilution massspectrometry was carried out with an internal standard. The followingmass transitions were used for detection and quantitation duringvalidation on positive polarity.

TABLE 1 Mass Transitions for Amyloid Beta fragments (Positive Polarity)Start Time End Time Precursor Collision Compound (min) (min) Polarity(m/z) Product (m/z) Energy (V) GAIIGLMVGGVV 0 10 Positive 1085.60 812.3731 GAIIGLMVGGVV 0 10 Positive 1085.60 869.40 33 GAIIGLMVGGVV 0 10Positive 1085.60 968.43 23 GAIIGLMVGGVVIA 0 10 Positive 1269.70 869.3943 GAIIGLMVGGVVIA 0 10 Positive 1269.70 968.44 34 GAIIGLMVGGVVIA 0 10Positive 1269.70 1067.50 32 GAIIGLMVGGVVIA 0 10 Positive 1269.70 1180.5723.8 IS 0 10 Positive 1110.70 768.48 35.5 IS 0 10 Positive 1110.70825.50 35.5 IS 0 10 Positive 1110.70 882.52 35.5

Example patient chromatograms are shown in FIGS. 6 and 7.

Various extractions were compared to optimize recovery of amyloid betafragments.

TABLE 2 Solid phase extraction recovery comparison Waters MAX Waters MCXWaters HLB Agilent C18 Phenomenex CX Thermo SAX Abeta 42 SPE Recovery 1181000 140244 125237 3869 86088 0 2 208763 153644 113481 4629 71027 0 3217163 153549 94726 2897 111434 0 4 238817 172517 131500 1662 92846 0MEAN 211435.75 154988.50 116236.00 3264.25 90348.75 0 SD 23916.6113273.14 16168.67 1281.97 16756.17 % CV 11.31% 8.56% 13.91% 39.27%18.55% Abeta 40 SPE Recovery 1 700570 517202 305741 18287 260704 0 2745439 534943 286890 27946 218579 0 3 824487 536441 244919 2660 365956 04 872219 637160 305132 16695 304490 0 MEAN 785678.75 556436.50 285670.5016397.00 287432.25 0 SD 77153.60 54520.39 28540.89 10420.26 63013.53 %CV 9.82% 9.80% 9.99% 63.55% 21.92% Waters MAX: mixed mode strong anionexchange → using for final assay validation Waters MXC: mixed modestrong cation exchange Waters HLB: silica made for hydrophobic compoundsAgilent C18: typical C18 packing Phenomenex CX: mixed mode strong cationexchange Thermo SAX: strong anion exchange

Mixed mode strong anion exchange provided the best recovery of amyloidbeta fragments.

Urea in the digestion increases recovery of analytes. The followingvalues were determined:

TABLE 3 Recovery comparison for urea vs. no urea Runs No Urea With UreaAB42 Recovery 1 15180 33397 2 14048 29445 3 17908 40026 4 14876 38011 516280 37141 MEAN 15778.00 36155.75 SD 1692.67 4634.14 % CV 10.73% 12.82%AB40 Recovery 1 95675 138525 2 97893 135433 3 121828 186864 4 99755157218 5 128836 167420 MEAN 112078.00 161733.75 SD 15588.07 21416.38 %CV 13.91% 13.24%

C4 analytical column was compared with C8 analytical column with respectto sensitivity. C4 column provided superior sensitivity, which isunexpected because C8 and C18 columns are generally preferred in theart. FIG. 11.

Example 4: Assay Reportable Range and Linearity

To establish the linearity of amyloid beta detection in the assay, oneblank assigned as zero standard and spiked standards were prepared andanalyzed. A quadratic regression from five consecutive runs yieldedcoefficient correlations of 0.995 or greater, with an accuracy of ±20%revealing a quantifiable linear range of 0.1 to 25 ng/mL. FIGS. 4 and 5.

Example 5: Assay Precision

Reproducibility of a sample within an assay was tested for the followingquality control levels:

Quality Control Beta Amyloid 40 Beta Amyloid 42 Level (pg/mL) (pg/mL)Low 750 750 Medium 7500 7500 High 15000 15000

Within Run Precision: Ten replicates of each quality control wereanalyzed within a single assay in the following order; low, medium andhigh.

Acceptability criteria: The % CV should be less than allowable ≤TEa/2.The TEa for this assay is determined to be 30%.

The % CV for Beta Amyloid 40 ranged from 14.31% to 6.55% across allthree quality control levels.

The % CV for Beta Amyloid 42 ranged from 14.93% to 7.51% across allthree quality control levels.

Total Precision: Data for inter-assay validation was analyzed.

Acceptability criteria: unacceptable if Total SD≥½ TEa or Total SD mustbe less than a defined maximum SD or CV.

The % CV should be less than allowable ≤TEa/2. The TEa for this assay isdetermined to be 30%.

The % CV for Beta Amyloid 40 ranged from 10.21% to 4.28% across allthree quality control levels.

The % CV for Beta Amyloid 42 ranged from 14.63% to 8.17% across allthree quality control levels.

Example 6: Analytical Sensitivity (Detection Limits)

Limit of Blank (LOB): Calculation: LOB=mean of blank+2SD.

Twenty-one matrix blanks were analyzed within a single assay, the backcalculated values are then used to calculate the LOB below.

LOB of AB40: 31.32 pg/mL.

LOB of AB42: 32.11 pg/mL.

AB 40 AB42  Run 1 22.85 3.20  Run 2 16.97 0.05  Run 3 14.62 15.31  Run 437.13 19.34  Run 5 −1.10 11.10  Run 6 50.10 5.28  Run 7 9.81 15.80  Run8 14.64 14.56  Run 9 10.31 12.74 Run 10 5.05 19.97 Run 11 1.96 25.07 Run12 30.81 18.98 Run 13 16.03 28.52 Run 14 8.93 22.49 Run 15 15.77 17.91Run 16 4.54 43.51 Run 17 −0.42 16.12 Run 18 28.03 48.73 Run 19 48.3636.87 Run 20 −4.17 17.58 Run 21 2.30 −13.54 MEAN 15.83 18.08 SD 15.4914.03 2D 30.97 28.07 LOB (Mean + 2D) 46.81 46.14

Limit of Detection (LOD): Calculation: LOD=mean of blank+4SD.

Twenty-one matrix blanks were analyzed within a single assay, the backcalculated values are then used to calculate the LOD below.

LOD of AB40: 77.78 pg/mL.

LOD of AB42: 74.21 pg/mL.

AB 40 AB42  Run 1 22.85 3.2  Run 2 16.97 0.05  Run 3 14.62 15.31  Run 437.13 19.34  Run 5 −1.1 11.1  Run 6 50.1 5.28  Run 7 9.81 15.8  Run 814.64 14.56  Run 9 10.31 12.74 Run 10 5.05 19.97 Run 11 1.96 25.07 Run12 30.81 18.98 Run 13 16.03 28.52 Run 14 8.93 22.49 Run 15 15.77 17.91Run 16 4.54 43.51 Run 17 −0.42 16.12 Run 18 28.03 48.73 Run 19 48.3636.87 Run 20 −4.17 17.58 Run 21 2.3 −13.54 MEAN 15.83 18.08 SD 15.4914.03 4D 61.95 56.14 LOD(Mean + 4D) 77.78 74.21

Limit of Quantitation (LOQ): Acceptability criteria: The lowestconcentration at which % CV is less than or equal to 2SD when TEa is30%.

The limit of quantitation for both Beta Amyloid 40 and 42 is determinedto be 100 pg/mL.

Example 7: Analyte Measurement Range (AMR)

Acceptability criteria: the average of the observed values shoulddeviate from the expected range by no more than 2SD or 20% CV when TEais 30%.

The % CV for Beta Amyloid 40 ranges from 6.52 to 12.01% which is deemedacceptable.

The % CV for Beta Amyloid 42 ranges from 6.27 to 14.24% which is deemedacceptable.

Beta Amyloid 40 Linearity pg/mL 100.00 250.00 500.00 1000.00 2500.005000.00 10000.00 25000.00 Oct. 30, 2015 85.05 283.27 535.17 937.012491.3 5123.81 9865.53 25029.13 Nov. 17, 2015 87.13 219.26 494 866.662025.84 4013.98 9356.08 23129.34 Nov. 18, 2015 89.26 265.71 408.57788.87 2162.14 3912.39 8316.37 19805.85 Nov. 19, 2015 108.91 268.39440.37 910.62 2052.88 4507.98 8741.97 20321.97 Nov. 20, 2015 106.77237.43 470.65 813.77 2077.58 4675.98 9212.27 21754.94 Nov. 21, 201585.59 237.05 423.71 790.07 1990.27 4854.98 9211.46 22913.33 MEAN 95.42254.81 469.75 863.39 2161.95 4446.83 9098.44 22008.25 STDev 11.46 25.8848.67 62.60 191.06 496.87 592.97 2129.60 % CV 12.01% 10.16% 10.36% 7.25%8.84% 11.17% 6.52% 9.68% % Accuracy 95.42% 101.92% 93.95% 86.34% 86.48%88.94% 90.98% 88.03% Beta Amyloid 42 Linearity pg/mL 100.00 250.00500.00 1000.00 2500.00 5000.00 10000.00 25000.00 Oct. 30, 2015 92.43264.26 496.06 1057.47 2574.61 4444.67 10573.78 24839.8 Nov. 17, 201585.02 303.85 530.61 943.81 2171.93 4542.56 10053.91 29125.77 Nov. 18,2015 103.46 238.3 440.25 1049.49 2484.67 5407.8 11219.31 26659.7 Nov.19, 2015 110.12 265.96 423.32 1069.96 2649.92 5431.65 11713.86 26435.43Nov. 20, 2015 120.9 299.3 417.11 1218.6 2873.72 6253.28 12913.3428399.67 Nov. 21, 2015 96.04 305.84 579.06 1129.86 2459.7 6134.4714436.49 29514.72 MEAN 102.39 274.33 461.47 1067.87 2550.97 5215.9911294.84 27092.07 STDev 14.18 27.22 49.62 98.18 256.22 742.92 1102.351698.28 % CV 13.85% 9.92% 10.75% 9.19% 10.04% 14.24% 9.76% 6.27% %Accuracy 102.39% 109.73% 92.29% 106.79% 102.04% 104.32% 112.95% 108.37%

Example 8: Accuracy

Recovery of known standards.

Acceptability criteria: the error due to lack of perfect recovery(amount recovered MINUS amount added) should be ≤2SD or 15% CV when TEais 30%.

Six stripped bovine cerebrospinal fluid samples were spiked at thefollowing concentrations: 1000, 2000, 3000, 4000, 8000, and 9000 pg/mL,each spike level was assayed in triplicate.

The six different spike levels were then diluted in 1 to 3 and 1 to 5ratios using stripped bovine cerebrospinal fluid as the diluent and alsoassayed in triplicate.

Pool 1 Beta Amyloid 40 pg/mL Baseline Spike Target ObservedConcentration Run 1 0.00 1000.00 838.66 Run 2 0.00 1000.00 962.28 Run 30.00 1000.00 809.68 MEAN 0.00 870.21 STDEV 0.00 81.04 % CV 0.00% 9.31% %Recovery 0.00% 87.02% Pool 2 Beta Amyloid 40 pg/mL Baseline Spike TargetObserved Concentration Run 1 8.83 2000.00 1915.62 Run 2 0.00 2000.001914.06 Run 3 9.04 2000.00 2053.31 MEAN 5.96 1961.00 STDEV 5.16 79.95 %CV 0.00% 4.08% % Recovery 0.60% 98.05% Pool 3 Beta Amyloid 40 pg/mLBaseline Spike Target Observed Concentration Run 1 0 3000 3170.69 Run 20 3000 3285.56 Run 3 0 3000 3565.91 MEAN 0.00 3340.72 STDEV 0.00 203.30% CV 0.00% 6.09% % Recovery 0.00% 111.36% Pool 4 Beta Amyloid 40 pg/mLBaseline Spike Target Observed Concentration Run 1 46.6 4000 4605.06 Run2 0 4000 4686.31 Run 3 10.18 4000 4740.77 MEAN 18.93 4677.38 STDEV 24.5068.29 % CV 0.00% 1.46% % Recovery 1.89% 116.93% Pool 5 Beta Amyloid 40pg/mL Baseline Spike Target Observed Concentration Run 1 14.11 70008051.86 Run 2 0 7000 8013.25 Run 3 4.41 7000 7968.89 MEAN 6.17 8011.33STDEV 7.22 41.52 % CV 0.00% 0.52% % Recovery 0.62% 100.14% Pool 6 BetaAmyloid 40 pg/mL Baseline Spike Target Observed Concentration Run 1 09000 9801.28 Run 2 6.5 9000 9152.91 Run 3 22.91 9000 9985.59 MEAN 9.809646.59 STDEV 11.81 437.36 % CV 0.00% 4.53% % Recovery 0.98% 107.18%

Pool 1 Beta Amyloid 40 pg/mL Spiked 1:3 Observed Concentration dilutiontarget 1:3 Concentration Run 1 838.66 279.553 254.25 Run 2 962.28320.760 262.19 Run 3 809.68 269.893 278.28 MEAN 870.21 290.07 264.91STDEV 81.04 12.24 % CV 0.00% 4.62% % Recovery 87.02% 91.33% Pool 2 BetaAmyloid 40 pg/mL 1:3 Observed dilution target 1:3 Concentration Run 11915.62 638.54 814.53 Run 2 1914.06 638.02 668.74 Run 3 2053.31 684.44740.84 MEAN 1961.00 653.67 741.37 STDEV 79.95 72.90 % CV 0.00% 9.83% %Recovery 98.05% 113.42% Pool 3 Beta Amyloid 40 pg/mL Spiked 1:3 ObservedConcentration dilution target 1:3 Concentration Run 1 3170.69 1056.901063.84 Run 2 3285.56 1095.19 1196.26 Run 3 3565.91 1188.64 1171.08 MEAN3340.72 1113.57 1143.73 STDEV 203.30 70.32 % CV 0.00% 6.15% % Recovery111.36% 102.71% Pool 4 Beta Amyloid 40 pg/mL Spiked 1:3 ObservedConcentration dilution target 1:3 Concentration Run 1 4605.06 1535.021660.34 Run 2 4686.31 1562.10 1601.44 Run 3 4740.77 1580.26 1761.25 MEAN4677.38 1559.13 1674.34 STDEV 68.29 80.82 % CV 0.00% 4.83% % Recovery116.93% 107.39% Pool 5 Beta Amyloid 40 pg/mL Spiked 1:3 ObservedConcentration dilution target 1:3 Concentration Run 1 8051.86 2683.952434.11 Run 2 8013.25 2671.08 2554.46 Run 3 7968.89 2656.30 2719.08 MEAN8011.33 2670.44 2569.22 STDEV 41.52 143.06 % CV 0.00% 5.57% % Recovery100.14% 96.21% Pool 6 Beta Amyloid 40 pg/mL Spiked 1:3 ObservedConcentration dilution target 1:3 Concentration Run 1 9801.28 3267.093594.42 Run 2 9152.91 3050.97 3230.93 Run 3 9985.59 3328.53 3183.79 MEAN9646.59 3215.53 3336.38 STDEV 437.36 224.71 % CV 0.00% 6.74% % Recovery107.18% 103.76%

Pool 1 Beta Amyloid 40 pg/mL Spiked 1:5 Observed Concentration dilutiontarget 1:5 Concentration Run 1 838.66 167.732 131.33 Run 2 962.28192.456 159.46 Run 3 809.68 161.936 153.29 MEAN 870.21 174.04 148.03STDEV 81.04 14.79 % CV 0.00% 9.99% % Recovery 87.02% 85.05% Pool 2 BetaAmyloid 40 pg/mL Spiked 1:5 Observed Concentration dilution target 1:5Concentration Run 1 1915.62 383.124 438.63 Run 2 1914.06 382.812 463.6Run 3 2053.31 410.662 365.06 MEAN 1961.00 392.20 422.43 STDEV 79.9551.23 % CV 0.00% 12.13% % Recovery 98.05% 107.71% Pool 3 Beta Amyloid 40pg/mL Spiked 1:5 Observed Concentration dilution target 1:5Concentration Run 1 3170.69 634.138 652.44 Run 2 3285.56 657.112 729.65Run 3 3565.91 713.182 719.26 MEAN 3340.72 668.14 700.45 STDEV 203.3041.90 % CV 0.00% 5.98% % Recovery 111.36% 104.84% Pool 4 Beta Amyloid 40pg/mL Spiked 1:5 Observed Concentration dilution target 1:5Concentration Run 1 4605.06 921.01 974.17 Run 2 4686.31 937.26 1007.06Run 3 4740.77 948.15 851.34 MEAN 4677.38 935.48 944.19 STDEV 68.29 82.07% CV 0.00% 8.69% % Recovery 116.93% 100.93% Pool 5 Beta Amyloid 40 pg/mLSpiked 1:5 Observed Concentration dilution target 1:5 Concentration Run1 8051.86 1610.37 1505.31 Run 2 8013.25 1602.65 1457.53 Run 3 7968.891593.78 1579.88 MEAN 8011.33 1602.27 1514.24 STDEV 41.52 61.66 % CV0.00% 4.07% % Recovery 100.14% 94.51% Pool 6 Beta Amyloid 40 pg/mLSpiked 1:5 Observed Concentration dilution target 1:5 Concentration Run1 9801.28 1960.26 1709.29 Run 2 9152.91 1830.58 1892.94 Run 3 9985.591997.12 1937.85 MEAN 9646.59 1929.32 1846.69 STDEV 437.36 121.09 % CV0.00% 6.56% % Recovery 107.18% 95.72%

Pool 1 Beta Amyloid 42 pg/mL Spike Observed Baseline TargetConcentration Run 1 90.26 1000.00 950.81 Run 2 108.39 1000.00 817.14 Run3 147.57 1000.00 1017.72 MEAN 115.41 928.56 STDEV 29.29 102.12 % CV0.00% 11.00% % Recovery 11.54% 92.86% Pool 2 Beta Amyloid 42 pg/mL SpikeObserved Baseline Target Concentration Run 1 25.38 2000.00 1638.08 Run 293.33 2000.00 1670.63 Run 3 39.62 2000.00 2228.58 MEAN 52.78 1845.76STDEV 35.83 331.93 % CV 67.90% 17.98% % Recovery 5.28% 92.29% Pool 3Beta Amyloid 42 pg/mL Spike Observed Baseline Target Concentration Run 171.92 3000 3487.59 Run 2 47.51 3000 3425.22 Run 3 45.29 3000 3393.22MEAN 54.91 3435.34 STDEV 14.78 47.99 % CV 26.91% 1.40% % Recovery 5.49%114.51% Pool 4 Beta Amyloid 42 pg/mL Spike Observed Baseline TargetConcentration Run 1 71.04 4000 4391.79 Run 2 151.45 4000 4753.15 Run 392.7 4000 4293.59 MEAN 105.06 4479.51 STDEV 41.61 242.01 % CV 39.60%5.40% % Recovery 10.51% 111.99% Pool 5 Beta Amyloid 42 pg/mL SpikeObserved Baseline Target Concentration Run 1 56.28 7000 7709.11 Run 2165.35 7000 7167.68 Run 3 85.57 7000 7688.12 MEAN 102.40 7521.64 STDEV56.45 306.72 % CV 55.13% 4.08% % Recovery 10.24% 107.45% Pool 6 BetaAmyloid 42 pg/mL Spike Observed Baseline Target Concentration Run 1103.51 9000 8046.03 Run 2 103.28 9000 7652.79 Run 3 67.54 9000 7382.95MEAN 91.44 7693.92 STDEV 20.70 333.45 % CV 22.64% 4.33% % Recovery 9.14%85.49%

Pool 1 Beta Amyloid 42 pg/mL Spiked 1:3 Observed Concentration dilutiontarget 1:3 Concentration Run 1 950.81 316.937 286.23 Run 2 817.14272.380 290.73 Run 3 1017.72 339.240 306.73 MEAN 928.56 309.52 294.56STDEV 102.12 10.77 % CV 0.00% 3.66% % Recovery 92.86% 95.17% Pool 2 BetaAmyloid 42 pg/mL 1:3 Observed dilution target 1:3 Concentration Run 11638.08 546.03 747.89 Run 2 1670.63 556.88 748.99 Run 3 2228.58 742.86688.05 MEAN 1845.76 615.25 728.31 STDEV 331.93 34.87 % CV 0.00% 4.79% %Recovery 184.58% 118.38% Pool 3 Beta Amyloid 42 pg/mL Spiked 1:3Observed Concentration dilution target 1:3 Concentration Run 1 3487.591162.53 1092.18 Run 2 3425.22 1141.74 1287.53 Run 3 3393.22 1131.071005.49 MEAN 3435.34 1145.11 1128.40 STDEV 47.99 144.47 % CV 0.00%12.80% % Recovery 343.53% 98.54% Pool 4 Beta Amyloid 42 pg/mL Spiked 1:3Observed Concentration dilution target 1:3 Concentration Run 1 4391.791463.93 1715.75 Run 2 4753.15 1584.38 1652.65 Run 3 4293.59 1431.201664.18 MEAN 4479.51 1493.17 1677.53 STDEV 242.01 33.60 % CV 0.00% 2.00%% Recovery 447.95% 112.35% Pool 5 Beta Amyloid 42 pg/mL Spiked 1:3Observed Concentration dilution target 1:3 Concentration Run 1 7709.112569.70 2112.71 Run 2 7167.68 2389.23 2256.35 Run 3 7688.12 2562.712377.18 MEAN 7521.64 2507.21 2248.75 STDEV 306.72 132.40 % CV 0.00%5.89% % Recovery 752.16% 89.69% Pool 6 Beta Amyloid 42 pg/mL Spiked 1:3Observed Concentration dilution target 1:3 Concentration Run 1 8046.032682.01 3205.47 Run 2 7652.79 2550.93 3178.28 Run 3 7382.95 2460.982754.6 MEAN 7693.92 2564.64 3046.12 STDEV 333.45 252.83 % CV 0.00% 8.30%% Recovery 769.39% 118.77%

Pool 1 Beta Amyloid 42 pg/mL Spiked 1:5 Observed Concentration dilutiontarget 1:5 Concentration Run 1 950.81 190.162 185.77 Run 2 817.14163.428 210.57 Run 3 1017.72 203.544 150.73 MEAN 928.56 185.71 182.36STDEV 102.12 30.07 % CV 0.00% 16.49% % Recovery 92.86% 98.19% Pool 2Beta Amyloid 42 pg/mL Spiked 1:5 Observed Concentration dilution target1:5 Concentration Run 1 1638.08 327.616 482.86 Run 2 1670.63 334.126415.91 Run 3 2228.58 445.716 407.04 MEAN 1845.76 369.15 435.27 STDEV331.93 41.45 % CV 0.00% 9.52% % Recovery 184.58% 117.91% Pool 3 BetaAmyloid 42 pg/mL Spiked 1:5 Observed Concentration dilution target 1:5Concentration Run 1 3487.59 697.518 832.7 Run 2 3425.22 685.044 686.42Run 3 3393.22 678.644 731.34 MEAN 3435.34 687.07 750.15 STDEV 47.9974.93 % CV 0.00% 9.99% % Recovery 343.53% 109.18% Pool 4 Beta Amyloid 42pg/mL Spiked 1:5 Observed Concentration dilution target 1:5Concentration Run 1 4391.79 878.36 875.68 Run 2 4753.15 950.63 1049.24Run 3 4293.59 858.72 936.27 MEAN 4479.51 895.90 953.73 STDEV 242.0188.09 % CV 0.00% 9.24% % Recovery 447.95% 106.45% Pool 5 Beta Amyloid 42pg/mL Spiked 1:5 Observed Concentration dilution target 1:5Concentration Run 1 7709.11 1541.82 1323.74 Run 2 7167.68 1433.541343.62 Run 3 7688.12 1537.62 1520.6 MEAN 7521.64 1504.33 1395.99 STDEV306.72 108.38 % CV 0.00% 7.76% % Recovery 752.16% 92.80% Pool 6 BetaAmyloid 42 pg/mL Spiked 1:5 Observed Concentration dilution target 1:5Concentration Run 1 8046.03 1609.21 1496.34 Run 2 7652.79 1530.561637.96 Run 3 7382.95 1476.59 1439.48 MEAN 7693.92 1538.78 1524.59 STDEV333.45 102.21 % CV 0.00% 6.70% % Recovery 769.39% 99.08%

Example 9: Specimen Stability

Acceptability criteria: A sample is considered stable as long as theaverage difference between the baseline value and the time/temperaturesample value is ≤TEa/2 for that analyte when TEa equals 30%.

Freeze/Thaw Stability: Freeze thaw analysis was conducted by analyzedsix patient pools which were divided into four even aliquots. All fouraliquots of each patient pool were frozen at −60 to −80° C. Aliquots twothrough four were thawed to ambient temperature of 18-26° C. and frozen.Aliquots three and four were thawed to ambient temperature of 18-26° C.and frozen. Aliquot four was then thawed to ambient temperature 18-26°C. and frozen.

All aliquots were thawed a final time to ambient temperature 18-26° C.and analyzed in technical triplicate. Freeze thaw analysis contains dataacross three freeze thaw cycles.

Beta Amyloid 40 and Beta Amyloid 42 have acceptable stability up to twofreeze thaw cycles.

Beta Amyloid 40 pg/mL Beta Amyloid 40 pg/mL Patient Pool 1 Patient Pool2 Baseline 1 FT 2 FT 3 FT Baseline 1 FT 2 FT 3 FT Run 1 9373.43 9530.9410628.69 11600.31 Run 1 11223.67 12887.34 13027.4 14939.98 Run 2 8513.869019.69 9464.10 11372.90 Run 2 11846.93 12478.66 12160.87 13997.01 Run 39761.12 9962.20 10505.74 9878.65 Run 3 12335.26 12993.07 13471.6413398.56 MEAN 9216.14 9504.28 10199.51 10950.62 MEAN 11801.95 12786.3612886.64 14111.85 STDEV 638.33 471.82 639.84 935.29 STDEV 557.16 271.67666.63 777.10 % CV 6.93% 4.96% 6.27% 8.54% % CV 4.72% 2.12% 5.17% 5.51%% Recovery 103.13% 110.67% 118.82% % Recovery 108.34% 109.19% 119.57%Total Mean 9967.64 Total Mean 12896.70 Total RSD 192.84 Total RSD 217.09Total % CV 6.68% Total % CV 4.38% Total % Recovery 110.87% Total %Recovery 112.37% Beta Amyloid 40 pg/mL Beta Amyloid 42 pg/mL PatientPool 1 Patient Pool 2 Baseline 1 FT 2 FT 3 FT Baseline 1 FT 2 FT 3 FTRun 1 1757.99 1957.06 1853.56 1368.85 Run 1 2091.28 2404.1 2882.042752.87 Run 2 1847.99 1999.14 1698.65 1802.71 Run 2 2838.46 3106.103110.09 2816.83 Run 3 1943.11 2227.49 1954.92 1741.27 Run 3 2826.063196.47 2911.49 2719.64 MEAN 1849.70 2061.23 1835.71 1637.61 MEAN2585.27 2902.22 2967.87 2763.11 STDEV 92.57 145.51 129.06 234.77 STDEV427.85 433.75 124.04 49.40 % CV 5.00% 7.06% 7.03% 14.34% % CV 16.55%14.95% 4.18% 1.79% % Recovery 111.44% 99.24% 88.53% % Recovery 112.26%114.80% 106.88% Total Mean 1846.06 Total Mean 2804.62 Total RSD 60.39Total RSD 200.99 Total % CV 8.36% Total % CV 9.37% Total % Recovery99.74% Total % Recovery 111.31% Beta Amyloid 40 pg/mL Beta Amyloid 40pg/mL Patient Pool 3 Patient Pool 4 Baseline 1 FT 2 FT 3 FT Baseline 1FT 2 FT 3 FT Run 1 13810.48 14226.23 16054.51 18419.42 Run 1 10459.4811019.24 12030.76 14514 Run 2 13737.02 14534.24 14958.05 17319.53 Run 210458.44 10590.22 11132.77 13398.93 Run 3 13395.15 14799.34 16085.0717381.51 Run 3 9700.34 10566.41 13475.75 11460.96 MEAN 13647.55 14519.9415699.21 17706.82 MEAN 10206.09 10725.29 12213.09 13124.63 STDEV 221.65286.82 642.05 617.91 STDEV 437.99 254.85 1182.08 1544.89 % CV 1.62%1.98% 4.09% 3.49% % CV 4.29% 2.38% 9.68% 11.77% % Recovery 106.39%115.03% 124.74% % Recovery 105.09% 119.66% 128.60% Total Mean 15393.38Total Mean 11567.28 Total RSD 218.78 Total RSD 610.20 Total % CV 2.79%Total % CV 7.03% Total % Recovery 117.06% Total % Recovery 117.78% BetaAmyloid 42 pg/mL Beta Amyloid 42 pg/mL Patient Pool 3 Patient Pool 4Baseline 1 FT 2 FT 3 FT Baseline 1 FT 2 FT Run 1 2211.66 2283.84 2641.041982.94 Run 1 1931.24 2499.50 2308.20 Run 2 2521.34 2687.66 2485.892457.18 Run 2 2771.11 2183.09 2418.84 Run 3 1849.56 2217.48 2504.982390.17 Run 3 2320.90 2260.08 1772.90 MEAN 2194.19 2396.33 2543.972276.76 MEAN 2341.08 2314.22 2166.65 STDEV 336.23 254.47 84.61 256.65STDEV 420.30 165.01 345.45 % CV 15.32% 10.62% 3.33% 11.27% % CV 17.95%7.13% 15.94% % Recovery 109.21% 115.94% 103.76% % Recovery 98.85% 92.55%Total Mean 2352.81 Total Mean 2273.98 Total RSD 105.98 Total RSD 131.24Total % CV 10.14% Total % CV 13.68% Total % Recovery 109.64% Total %Recovery 95.70% Beta Amyloid 40 pg/mL Beta Amyloid 40 pg/mL Patient Pool5 Patient Pool 6 Baseline 1 FT 2 FT 3 FT Baseline 1 FT 2 FT 3 FT Run 110636.38 11289.21 12568.68 14488.23 Run 1 10743.46 11195.73 12151.7712151.77 Run 2 10219.63 10610.52 11752.49 14421 Run 2 9924.85 10721.2412368.51 12368.51 Run 3 10973.86 10517.58 13479.79 12081.94 Run 311094.19 10635.12 12526.26 13027.58 MEAN 10609.96 10805.77 12600.3213663.72 MEAN 10587.50 10850.70 12348.85 12515.95 STDEV 377.81 421.24864.08 1370.28 STDEV 600.07 301.89 188.02 456.14 % CV 3.56% 3.90% 6.86%10.03% % CV 5.67% 2.78% 1.52% 3.64% % Recovery 101.85% 118.76% 128.78% %Recovery 102.49% 116.64% 118.21% Total Mean 11919.94 Total Mean 11575.75Total RSD 463.35 Total RSD 179.83 Total % CV 6.09% Total % CV 3.40%Total % Recovery 116.46% Total % Recovery 112.45% Beta Amyloid 42 pg/mLBeta Amyloid 42 pg/mL Patient Pool 5 Patient Pool 6 Baseline 1 FT 2 FT 3FT Baseline 1 FT 2 FT 3 FT Run 1 2126.51 2246.83 2749.21 2501.28 Run 12027.03 1966.27 2348.20 2236.04 Run 2 2423.15 2325.82 2979.89 2698.43Run 2 2179.57 2086.16 2302.52 2256.19 Run 3 2937.29 2138.08 2621.432671.59 Run 3 2579.21 2128.10 2094.63 1809.31 MEAN 2495.65 2236.912783.51 2623.77 MEAN 2261.94 2060.18 2248.45 2100.51 STDEV 410.22 94.26181.67 106.92 STDEV 285.16 83.99 135.16 252.39 % CV 16.44% 4.21% 6.53%4.08% % CV 12.61% 4.08% 6.01% 12.02% % Recovery 89.63% 111.53% 105.13% %Recovery 91.08% 99.40% 92.86% Total Mean 2534.96 Total Mean 2167.77Total RSD 146.47 Total RSD 95.20 Total % CV 7.81% Total % CV 8.68% Total% Recovery 102.10% Total % Recovery 94.45%

Extracted Sample Stability: Ten samples were analyzed the same day assample extraction for a baseline value. The next day, the same sampleswere re-injected for analysis against the baseline values.

Beta Amyloid 40 and 42 shows extracted sample stability up to 1 day at4° C. in the C-stack of the CTC Autosampler.

Beta Amyloid 40 pg/mL Baseline 1 day MEAN SD % CV % Recovery Sample 10.10 0.11 0.11 0.01 6.73% 110.00% Sample 2 0.21 0.22 0.22 0.01 3.29%104.76% Sample 3 0.49 0.52 0.51 0.02 4.20% 106.12% Sample 4 1.17 1.071.12 0.07 6.31% 91.45% Sample 5 2.90 2.86 2.88 0.03 0.98% 98.62% Sample6 4.62 5.20 4.91 0.41 8.35% 112.55% Sample 7 25.07 25.37 25.22 0.210.84% 101.20% Sample 8 0.80 0.76 0.78 0.03 3.63% 95.00% Sample 9 8.178.02 8.10 0.11 1.31% 98.16% Sample 10 16.24 14.33 15.29 1.35 8.84%88.24%

Refrigerated Stability (2.0 to 8.0° C.): Samples have shown to be stablerefrigerated for up to 5 days.

POOL 1 Beta Amyloid 40 600 pg/mL Replicate 1 Replicate 2 Replicate 2MEAN SD % CV % Recovery Baseline 779.29 738.39 583.39 700.36 103.3414.76% 116.73% Day 1 658.23 659.68 776.3 698.07 67.75 9.71% 116.35% Day3 767.06 713.26 658.66 712.99 54.20 7.60% 118.83% Day 5 678.63 749.03649.66 692.44 51.10 7.38% 115.41% Day 7 693.77 703.42 733.24 710.1420.58 2.90% 118.36% Day 14 946.19 831.81 730.22 836.07 108.05 12.92%139.35% Day 21 599.04 608.87 678.33 628.75 43.22 6.87% 104.79% Day 31640.92 745.31 741.69 709.31 59.25 8.35% 118.22% POOL 2 Beta Amyloid 403000 pg/mL Replicate 1 Replicate 2 Replicate 2 MEAN SD % CV % RecoveryBaseline 3095.88 2786.21 2749.96 2877.35 190.12 6.61% 95.91% Day 13088.14 3270.74 2975.15 3111.34 149.15 4.79% 103.71% Day 3 2673.93470.73 3293.98 3146.20 418.47 13.30% 104.87% Day 5 2965.85 2791.083209.31 2988.75 210.05 7.03% 99.62% Day 7 3019.89 3405.5 3197.44 3207.61193.01 6.02% 106.92% Day 14 3112.61 2942.44 2878.28 2977.78 121.10 4.07%99.26% Day 21 3339.29 3159.82 2795.68 3098.26 276.98 8.94% 103.28% Day31 3063.34 3238.69 2775.26 3025.76 233.99 7.73% 100.86% POOL 3 BetaAmyloid 40 pg/mL 9000 pg/mL Replicate 1 Replicate 2 Replicate 2 MEAN SD% CV % Recovery Baseline 8599.60 9082.10 9287.20 8989.63 353.00 3.93%99.88% Day 1 8136.03 8785.38 7959.96 8293.79 434.74 5.24% 92.15% Day 38802.53 10914.81 8954.65 9557.33 1178.07 12.33% 106.19% Day 5 8703.299246.50 8335.09 8761.63 458.50 5.23% 97.35% Day 7 7697.84 9428.178620.20 8582.07 865.79 10.09% 95.36% Day 14 8247.15 8505.88 8240.758331.26 151.26 1.82% 92.57% Day 21 8162.27 8182.13 7900.43 8081.61157.22 1.95% 89.80% Day 31 7765.88 8581.78 8139.16 8162.27 408.44 5.00%90.69% POOL 4 Beta Amyloid 40 pg/mL 1200 pg/mL Replicate 1 Replicate 2Replicate 2 MEAN SD % CV % Recovery Baseline 1083.17 1007.45 1237.131109.25 117.04 10.55% 92.44% Day 1 1019.62 1283.73 1116.96 1140.10133.57 11.72% 95.01% Day 3 1216.89 1249.03 1062.99 1176.30 99.44 8.45%98.03% Day 5 1129.71 1132.32 1084.54 1115.52 26.86 2.41% 92.96% Day 71071.05 1096.81 1101.76 1089.87 16.49 1.51% 90.82% Day 14 993.74 1148.191065.45 1069.13 77.29 7.23% 89.09% Day 21 1153.51 1150.47 1201.111168.36 28.40 2.43% 97.36% Day 31 1146.57 1141.36 999.53 1095.82 83.437.61% 91.32% POOL 5 Beta Amyloid 40 pg/mL 6000 pg/mL Replicate 1Replicate 2 Replicate 2 MEAN SD % CV % Recovery Baseline 6677.96 6380.356933.19 6663.83 276.69 4.15% 111.06% Day 1 5355.05 5489.45 6317.585720.69 521.27 9.11% 95.34% Day 3 5620.59 6561.63 6009.07 6063.76 472.907.80% 101.06% Day 5 5395.23 5481.01 5842.92 5573.05 237.61 4.26% 92.88%Day 7 5555.9 5810.19 5689.14 5685.08 127.19 2.24% 94.75% Day 14 6075.725873.62 5753.26 5900.87 162.95 2.76% 98.35% Day 21 5702.75 6152.045739.07 5864.62 249.57 4.26% 97.74% Day 31 5495.09 6234.34 5814.45847.94 370.76 6.34% 97.47% POOL 6 Beta Amyloid 40 20000 pg/mL Replicate1 Replicate 2 Replicate 2 MEAN SD % CV % Recovery Baseline 20310.4520876.37 21447.84 20878.22 568.70 2.72% 104.39% Day 1 17537.07 19362.3419307.88 18735.76 1038.46 5.54% 93.68% Day 3 18592.96 19095.83 20956.0919548.29 1244.84 6.37% 97.74% Day 5 17915.26 17751.53 18930.57 18199.12638.72 3.51% 91.00% Day 7 19548.87 18232.47 18022.25 18601.20 827.414.45% 93.01% Day 14 16869.19 18533.17 18083.45 17828.60 860.77 4.83%89.14% Day 21 18377.09 18761.43 19195.86 18778.13 409.64 2.18% 93.89%Day 31 18050.69 18667.82 18603.41 18440.64 339.24 1.84% 92.20%

POOL 1 Beta Amyloid 42 600 pg/mL Replicate 1 Replicate 2 Replicate 2MEAN SD % CV % Recovery Baseline 715.19 711.63 597.55 674.79 66.92 9.92%112.47% Day 1 637.32 664.25 592.10 631.22 36.46 5.78% 105.20% Day 3637.32 681.03 640.01 652.79 24.50 3.75% 108.80% Day 5 521.06 655.35641.83 606.08 73.94 12.20% 101.01% Day 7 548.16 688.76 694.50 643.8182.88 12.87% 107.30% Day 14 793.24 708.37 745.63 749.08 42.54 5.68%124.85% Day 21 456.42 530.45 520.18 502.35 40.11 7.98% 83.73% Day 31596.89 729.56 636.46 654.30 68.11 10.41% 109.05% POOL 2 Beta Amyloid 423000 pg/mL Replicate 1 Replicate 2 Replicate 2 MEAN SD % CV % RecoveryBaseline 2948.02 2894.12 3253.65 3031.93 193.90 6.40% 101.06% Day 12362.8 2918.64 2765.25 2682.23 287.07 10.70% 89.41% Day 3 2511.772628.37 2544.33 2561.49 60.16 2.35% 85.38% Day 5 2815.34 2710.19 2739.482755.00 54.27 1.97% 91.83% Day 7 2214.73 2551.56 2602.08 2456.12 210.578.57% 81.87% Day 14 2005.43 1967.07 2122.07 2031.52 80.73 3.97% 67.72%Day 21 2925.33 2407.52 2575.49 2636.11 264.17 10.02% 87.87% Day 311887.95 2061.06 2050.62 1999.88 97.07 4.85% 66.66% POOL 3 Beta Amyloid42 pg/mL 9000 pg/mL Replicate 1 Replicate 2 Replicate 2 MEAN SD % CV %Recovery Baseline 7683.62 7786.05 7986.82 7818.83 154.24 1.97% 86.88%Day 1 7746.48 6971.53 9102.61 7940.21 1078.67 13.58% 88.22% Day 37416.17 8997.58 7465.98 7959.91 898.99 11.29% 88.44% Day 5 7505.897789.58 7599.32 7631.60 144.57 1.89% 84.80% Day 7 5685.59 6813.6 5857.26118.80 607.80 9.93% 67.99% Day 14 4331.85 4542.49 4939.12 4604.49308.35 6.70% 51.16% Day 21 4221.04 4897.48 4225.05 4447.86 389.39 8.75%49.42% Day 31 3865.69 5027.86 4997.76 4630.44 662.46 14.31% 51.45% POOL4 Beta Amyloid 42 pg/mL 1200 pg/mL Replicate 1 Replicate 2 Replicate 2MEAN SD % CV % Recovery Baseline 1083.17 1007.45 1237.13 1109.25 117.0410.55% 92.44% Day 1 1046.48 1215.49 1074.39 1112.12 90.60 8.15% 92.68%Day 3 1045.3 1119.79 1318.35 1161.15 141.14 12.16% 96.76% Day 5 1032.691172.59 1374.1 1193.13 171.63 14.38% 99.43% Day 7 968.59 1192.34 1327.841162.92 181.42 15.60% 96.91% Day 14 1102.62 1156.67 1243.97 1167.7571.32 6.11% 97.31% Day 21 1380.18 1179.06 1165.8 1241.68 120.13 9.67%103.47% Day 31 1341.91 1445.29 1491.16 1426.12 76.45 5.36% 118.84% POOL5 Beta Amyloid 42 pg/mL 6000 pg/mL Replicate 1 Replicate 2 Replicate 2MEAN SD % CV % Recovery Baseline 6677.96 6380.35 6933.19 6663.83 276.694.15% 111.06% Day 1 5571.06 6471.21 6952.26 6331.51 701.12 11.07%105.53% Day 3 5474.18 6588.7 6046.94 6036.61 557.33 9.23% 100.61% Day 56496.2 6827.73 7163.7 6829.21 333.75 4.89% 113.82% Day 7 5616.75 6227.286454.22 6099.42 433.13 7.10% 101.66% Day 14 5145.26 6335.6 7603.786361.55 1229.47 19.33% 106.03% Day 21 5272.62 5451.41 5770.77 5498.27252.36 4.59% 91.64% Day 31 4507.56 5739.15 6495.96 5580.89 1003.6017.98% 93.01% POOL 6 Beta Amyloid 42 20000 pg/mL Replicate 1 Replicate 2Replicate 2 MEAN SD % CV % Recovery Baseline 21756.95 21339.04 20496.0521197.35 642.28 3.03% 105.99% Day 1 18082.02 17251.16 19283.78 18205.651021.93 5.61% 91.03% Day 3 17597.95 16670.16 19199.17 17822.43 1279.367.18% 89.11% Day 5 19385.22 18263.81 15284.42 17644.48 2119.39 12.01%88.22% Day 7 14779.5 16218.56 16987.23 15995.10 1120.70 7.01% 79.98% Day14 15267.66 15004.07 16492.44 15588.06 794.23 5.10% 77.94% Day 2114152.8 16347.63 18450.29 16316.91 2148.91 13.17% 81.58% Day 31 15242.6515307.45 16358.04 15636.05 626.10 4.00% 78.18%

Room Temperature Stability (18.0 to 26.0° C.): Samples are stable up to3 days at 18-26° C.

POOL 1 Beta Amyloid 40 600 pg/mL Replicate 1 Replicate 2 Replicate 2MEAN SD % CV % Recovery Baseline 779.29 738.39 583.39 700.36 103.3414.76% 116.73% Day 1 597.97 555.18 651.41 601.52 48.21 8.02% 100.25% Day3 677.29 639.59 627.46 648.11 25.99 4.01% 108.02% Day 5 483.68 489.69435.26 469.54 29.84 6.36% 78.26% Day 7 20.19 7.97 22.45 16.87 7.7946.18% 2.81% Day 14 41.15 6.67 46.31 31.38 21.55 68.69% 5.23% Day 214.64 28.6 36.02 23.09 16.40 71.04% 3.85% Day 31 19.96 0.88 40.91 20.5820.02 97.27% 3.43% POOL 2 Beta Amyloid 40 3000 pg/mL Replicate 1Replicate 2 Replicate 2 MEAN SD % CV % Recovery Baseline 3073.58 1501.463340.37 2638.47 993.67 37.66% 87.95% Day 1 2648.58 3019.2 2365.832677.87 327.67 12.24% 89.26% Day 3 3170.96 3122.42 2934.33 3075.90124.99 4.06% 102.53% Day 5 2780.11 2791.29 3224.36 2931.92 253.32 8.64%97.73% Day 7 2623.02 2768.02 3080.48 2823.84 233.78 8.28% 94.13% Day 14256.08 308.73 267.08 277.30 27.77 10.02% 9.24% Day 21 14.05 25.17 22.3220.51 5.78 28.16% 0.68% Day 31 43.41 39.32 29.48 37.40 7.16 19.14% 1.25%POOL 3 Beta Amyloid 40 pg/mL 9000 pg/mL Replicate 1 Replicate 2Replicate 2 MEAN SD % CV % Recovery Baseline 8599.60 9082.10 9287.208989.63 353.00 3.93% 99.88% Day 1 8064.65 10059.63 9271.61 9131.961004.79 11.00% 101.47% Day 3 7135.90 7190.06 7525.27 7283.74 210.912.90% 80.93% Day 5 370.90 287.98 337.43 332.10 41.72 12.56% 3.69% Day 72691.50 2768.60 2806.56 2755.55 58.63 2.13% 30.62% Day 14 346.01 367.40466.41 393.27 64.23 16.33% 4.37% Day 21 625.28 824.15 719.24 722.8999.49 13.76% 8.03% Day 31 1638.52 520.62 1844.42 1334.52 712.34 53.38%14.83% POOL 4 Beta Amyloid 40 pg/mL 1200 pg/mL Replicate 1 Replicate 2Replicate 2 MEAN SD % CV % Recovery Baseline 1147.09 1303.82 1184.021211.64 81.94 6.76% 100.97% Day 1 1195.71 1252.52 1293 1247.08 48.873.92% 103.92% Day 3 1133.27 1280.53 1194.55 1202.78 73.97 6.15% 100.23%Day 5 1148.79 1287.75 1100.57 1179.04 97.19 8.24% 98.25% Day 7 1069.39968.4 1104.36 1047.38 70.60 6.74% 87.28% Day 14 0 120.26 0 40.09 69.43173.21% 3.34% Day 21 16.71 32.4 34.8 27.97 9.83 35.13% 2.33% Day 31143.94 −4.32 124.87 88.16 80.66 91.49% 7.35% POOL 5 Beta Amyloid 40pg/mL 6000 pg/mL Replicate 1 Replicate 2 Replicate 2 MEAN SD % CV %Recovery Baseline 6542.39 6115.93 6015.49 6224.60 279.76 4.49% 103.74%Day 1 6245.93 6475.35 6066.35 6262.54 205.01 3.27% 104.38% Day 3 6639.616173.74 6044.22 6285.86 313.13 4.98% 104.76% Day 5 6285.39 6701.625615.48 6200.83 547.99 8.84% 103.35% Day 7 5047.34 4152.02 4746.154648.50 455.58 9.80% 77.48% Day 14 4196.56 4495.05 4094.12 4261.91208.30 4.89% 71.03% Day 21 300.12 417.39 350.55 356.02 58.83 16.52%5.93% Day 31 43.23 70.37 192.66 102.09 79.60 77.98% 1.70% POOL 6 BetaAmyloid 40 20000 pg/mL Replicate 1 Replicate 2 Replicate 2 MEAN SD % CV% Recovery Baseline 20310.45 20876.37 21447.84 20878.22 568.70 2.72%104.39% Day 1 21141.19 22951.71 19538.39 21210.43 1707.71 8.05% 106.05%Day 3 19831.93 21044.04 20583.6 20486.52 611.86 2.99% 102.43% Day 519857.52 21786.11 19913.92 20519.18 1097.55 5.35% 102.60% Day 7 7033.677265.53 7245.05 7181.42 128.36 1.79% 35.91% Day 14 592.23 821.65 2138.351184.08 834.35 70.46% 5.92% Day 21 2417.51 2478.89 2553.32 2483.24 68.012.74% 12.42% Day 31 9604.6 10638.74 10448.29 10230.54 550.38 5.38%51.15%

POOL 1 Beta Amyloid 42 600 pg/mL Replicate 1 Replicate 2 Replicate 2MEAN SD % CV % Recovery Baseline 715.19 711.63 597.55 674.79 66.92 9.92%112.47% Day 1 707.17 691.20 635.20 677.86 37.79 5.58% 112.98% Day 3713.79 601.45 724.24 679.83 68.08 10.01% 113.30% Day 5 623.04 722.61535.83 627.16 93.46 14.90% 104.53% Day 7 86.65 67.82 98.02 84.16 15.2518.12% 14.03% Day 14 148.17 147.30 198.27 164.58 29.18 17.73% 27.43% Day21 1221.34 1287.15 1392.07 1300.19 86.11 6.62% 216.70% Day 31 21.71100.48 82.78 68.32 41.33 60.49% 11.39% POOL 2 Beta Amyloid 42 3000 pg/mLReplicate 1 Replicate 2 Replicate 2 MEAN SD % CV % Recovery Baseline2861.62 2890.83 3308.1 3020.18 249.77 8.27% 100.67% Day 1 2579.3 2366.573060.43 2668.77 355.48 13.32% 88.96% Day 3 3060.43 3106.46 3072.93079.93 23.81 0.77% 102.66% Day 5 2340.81 2751.9 2688.63 2593.78 221.358.53% 86.46% Day 7 2766.13 2542.61 2929.77 2746.17 194.35 7.08% 91.54%Day 14 505.01 593.27 685.43 594.57 90.22 15.17% 19.82% Day 21 321.55375.87 334.76 344.06 28.33 8.23% 11.47% Day 31 172.5 205.61 308.62228.91 70.99 31.01% 7.63% POOL 3 Beta Amyloid 42 pg/mL 9000 pg/mLReplicate 1 Replicate 2 Replicate 2 MEAN SD % CV % Recovery Baseline7683.62 7786.05 7986.82 7818.83 154.24 1.97% 86.88% Day 1 7101.318620.92 7680.41 7800.88 766.93 9.83% 86.68% Day 3 6201.38 6152.6 7085.816479.93 525.27 8.11% 72.00% Day 5 1220.28 1539.99 1209.82 1323.36 187.6814.18% 14.70% Day 7 2878.85 2559.36 2916.04 2784.75 196.08 7.04% 30.94%Day 14 1108.22 1249.35 988.14 1115.24 130.75 11.72% 12.39% Day 21 187.09409.35 541.69 379.38 179.19 47.23% 4.22% Day 31 730.47 741.9 500.54657.64 136.17 20.71% 7.31% POOL 4 Beta Amyloid 42 pg/mL 1200 pg/mLReplicate 1 Replicate 2 Replicate 2 MEAN SD % CV % Recovery Baseline1334.21 1203.74 1451.67 1329.87 124.02 9.33% 110.82% Day 1 1022.21328.08 1184.74 1178.34 153.04 12.99% 98.20% Day 3 1186.53 1093.621293.39 1191.18 99.97 8.39% 99.27% Day 5 1021.25 1468.26 1030.04 1173.18255.58 21.79% 97.77% Day 7 1046.77 1055.15 1071.49 1057.80 12.57 1.19%88.15% Day 14 831.31 579.26 1369.95 926.84 403.91 43.58% 77.24% Day 21152.52 397.68 233.18 261.13 124.95 47.85% 21.76% Day 31 760.66 151.54258.9 390.37 325.15 83.29% 32.53% POOL 5 Beta Amyloid 42 pg/mL 6000pg/mL Replicate 1 Replicate 2 Replicate 2 MEAN SD % CV % RecoveryBaseline 6821.3 6333.16 6095.21 6416.56 370.16 5.77% 106.94% Day 15607.25 7000.01 6326.05 6311.10 696.50 11.04% 105.19% Day 3 6966.145878.18 6083.74 6309.35 578.01 9.16% 105.16% Day 5 5571.6 6456.155511.17 5846.31 529.00 9.05% 97.44% Day 7 5713.91 4808.07 5766.285429.42 538.74 9.92% 90.49% Day 14 4462.05 4518.25 3740.19 4240.16433.90 10.23% 70.67% Day 21 437.82 401.52 542.97 460.77 73.46 15.94%7.68% Day 31 600.29 617.72 865.64 694.55 148.42 21.37% 11.58% POOL 6Beta Amyloid 42 20000 pg/mL Replicate 1 Replicate 2 Replicate 2 MEAN SD% CV % Recovery Baseline 21756.95 21339.04 20496.05 21197.35 642.283.03% 105.99% Day 1 22016.05 21569.11 19770.44 21118.53 1188.68 5.63%105.59% Day 3 21983.38 23840.4 21233.98 22352.59 1341.86 6.00% 111.76%Day 5 20061.84 19555.29 20219.32 19945.48 346.97 1.74% 99.73% Day 78155.77 9523.15 9885.37 9188.10 912.18 9.93% 45.94% Day 14 3224.14504.06 6381.28 4703.15 1587.98 33.76% 23.52% Day 21 2571.06 2533.153191.43 2765.21 369.60 13.37% 13.83% Day 31 4629.03 4807.18 3974.244470.15 438.61 9.81% 22.35%

Frozen Stability (−10.0 to −30.0° C.): Samples are stable up to 31 daysat −10 to −30° C.

POOL 1 Beta Amyloid 40 600 pg/mL Replicate 1 Replicate 2 Replicate 2MEAN SD % CV % Recovery Baseline 646.34 630.12 623.37 633.28 11.81 1.86%105.55% Day 1 553.74 649.95 737.08 646.92 91.71 14.18% 107.82% Day 3679.57 717.08 645.37 680.67 35.87 5.27% 113.45% Day 5 572.02 681.43629.8 627.75 54.73 8.72% 104.63% Day 7 543.13 642.62 649.39 611.71 59.499.73% 101.95% Day 14 596.61 636.47 561.51 598.20 37.51 6.27% 99.70% Day21 612.82 585.02 586.71 594.85 15.59 2.62% 99.14% Day 31 586.31 596.06677.97 620.11 50.34 8.12% 103.35% POOL 2 Beta Amyloid 40 3000 pg/mLReplicate 1 Replicate 2 Replicate 2 MEAN SD % CV % Recovery Baseline3095.88 2786.21 2749.96 2877.35 190.12 6.61% 95.91% Day 1 3212.213465.63 3022.97 3233.60 222.10 6.87% 107.79% Day 3 2895.18 2980.172984.49 2953.28 50.36 1.71% 98.44% Day 5 2825.26 3536.06 3441.65 3267.66386.02 11.81% 108.92% Day 7 3166.74 2846.27 2954.38 2989.13 163.04 5.45%99.64% Day 14 2511.23 2775.96 3020.4 2769.20 254.65 9.20% 92.31% Day 212511.23 2775.96 3020.4 2769.20 254.65 9.20% 92.31% Day 31 2893.27 3086.62854.03 2944.63 124.50 4.23% 98.15% POOL 3 Beta Amyloid 40 9000 pg/mLReplicate 1 Replicate 2 Replicate 2 MEAN SD % CV % Recovery Baseline9041.95 8204.27 9240.21 8828.81 549.88 6.23% 98.10% Day 1 8783.710827.22 8982.54 9531.15 1126.82 11.82% 105.90% Day 3 9211.93 9149.868867.29 9076.36 183.70 2.02% 100.85% Day 5 9182.12 9997.19 9886.459688.59 442.09 4.56% 107.65% Day 7 8569.08 8999.58 9511.47 9026.71471.78 5.23% 100.30% Day 14 9220.4 9028.98 8064.85 8771.41 619.34 7.06%97.46% Day 21 8424.04 9700.88 9775.48 9300.13 759.64 8.17% 103.33% Day31 8912.04 7891.96 8515.74 8439.91 514.25 6.09% 93.78% POOL 4 BetaAmyloid 40 6000 pg/mL Replicate 1 Replicate 2 Replicate 2 MEAN SD % CV %Recovery Baseline 6453.76 5952.82 5980.68 6129.09 281.52 4.59% 102.15%Day 1 6614.06 6027.79 6375.06 6338.97 294.80 4.65% 105.65% Day 3 5756.916360.45 6702.4 6273.25 478.74 7.63% 104.55% Day 5 5726.32 5715.8 7001.746147.95 739.42 12.03% 102.47% Day 7 5126.17 6209.65 6422.83 5919.55695.31 11.75% 98.66% Day 14 5598.62 5514.06 5780.43 5631.04 136.11 2.42%93.85% Day 21 5780.43 7030.06 6223.43 6344.64 633.57 9.99% 105.74% Day31 6861.57 6840.4 6010.14 6570.70 485.58 7.39% 109.51% POOL 5 BetaAmyloid 40 1200 pg/mL Replicate 1 Replicate 2 Replicate 2 MEAN SD % CV %Recovery Baseline 1310.92 1162.27 1209.75 1227.65 75.92 6.18% 102.30%Day 1 1241.58 1149.45 1391.02 1260.68 121.91 9.67% 105.06% Day 3 1076.871083.95 1027.59 1062.80 30.70 2.89% 88.57% Day 5 1132.64 1284.34 1234.741217.24 77.35 6.35% 101.44% Day 7 1291.93 1451.69 1315.11 1352.91 86.336.38% 112.74% Day 14 1172.05 1203.56 1232.12 1202.58 30.05 2.50% 100.21%Day 21 1241.21 1110.38 1168.52 1173.37 65.55 5.59% 97.78% Day 31 1083.951027.59 1156.08 1089.21 64.41 5.91% 90.77%

POOL 1 Beta Amyloid 42 600 pg/mL Replicate 1 Replicate 2 Replicate 2MEAN SD % CV % Recovery Baseline 551.44 624.68 669.50 615.21 59.60 9.69%102.53% Day 1 564.89 622.57 633.74 607.07 36.95 6.09% 101.18% Day 3525.73 459.44 576.28 520.48 58.60 11.26% 86.75% Day 5 648.20 753.93567.60 656.58 93.45 14.23% 109.43% Day 7 509.36 559.91 615.35 561.5453.01 9.44% 93.59% Day 14 466.70 632.30 618.20 572.40 91.81 16.04%95.40% Day 21 574.67 447.54 556.89 526.37 68.84 13.08% 87.73% Day 31466.33 493.23 583.68 514.41 61.48 11.95% 85.74% POOL 2 Beta Amyloid 423000 pg/mL Replicate 1 Replicate 2 Replicate 2 MEAN SD % CV % RecoveryBaseline 2948.02 2894.12 3253.65 3031.93 193.90 6.40% 101.06% Day 13329.08 2842.89 3721.36 3297.78 440.07 13.34% 109.93% Day 3 3070.813338.41 3281.97 3230.40 141.06 4.37% 107.68% Day 5 3162.79 3783.343701.06 3549.06 337.04 9.50% 118.30% Day 7 3009.29 2966.92 3399.73125.30 238.58 7.63% 104.18% Day 14 2648.36 2957.23 3382 2995.86 368.3412.30% 99.86% Day 21 2648.36 2957.23 3382 2995.86 368.34 12.30% 99.86%Day 31 3695.08 3249.83 3065.84 3336.92 323.53 9.70% 111.23% POOL 3 BetaAmyloid 42 9000 pg/mL Replicate 1 Replicate 2 Replicate 2 MEAN SD % CV %Recovery Baseline 9131.88 8396.59 8877.05 8801.84 373.37 4.24% 97.80%Day 1 10014.67 11304.49 9468.86 10262.67 942.61 9.18% 114.03% Day 38053.3 9772.46 9749.97 9191.91 986.13 10.73% 102.13% Day 5 8475.8810403.45 10578.46 9819.26 1166.69 11.88% 109.10% Day 7 8441.49 9248.8910118.42 9269.60 838.66 9.05% 103.00% Day 14 9507.55 9268.16 8255.549010.42 664.61 7.38% 100.12% Day 21 9372.95 10354.65 9516.11 9747.90530.31 5.44% 108.31% Day 31 9454.97 9227.46 8902.68 9195.04 277.57 3.02%102.17% POOL 4 Beta Amyloid 42 6000 pg/mL Replicate 1 Replicate 2Replicate 2 MEAN SD % CV % Recovery Baseline 6677.96 6380.35 6933.196663.83 276.69 4.15% 111.06% Day 1 5985.48 5498.94 6791.57 6092.00652.86 10.72% 101.53% Day 3 5446.16 6668.14 7105.19 6406.50 859.9113.42% 106.77% Day 5 5870.58 5233.08 6853.32 5985.66 816.23 13.64%99.76% Day 7 5703.01 5989.02 6362.57 6018.20 330.75 5.50% 100.30% Day 145834.97 6306.89 5534.01 5891.96 389.58 6.61% 98.20% Day 21 5534.017308.97 6528.39 6457.12 889.62 13.78% 107.62% Day 31 6305.84 7144.755356.93 6269.17 894.47 14.27% 104.49% POOL 5 Beta Amyloid 42 1200 pg/mLReplicate 1 Replicate 2 Replicate 2 MEAN SD % CV % Recovery Baseline1083.17 1007.45 1237.13 1109.25 117.04 10.55% 92.44% Day 1 1322.221270.31 1082.88 1225.14 125.90 10.28% 102.09% Day 3 1024.85 949.35 907.3960.50 59.56 6.20% 80.04% Day 5 1249.41 971.58 1231.45 1150.81 155.4813.51% 95.90% Day 7 1053.04 1275.89 1263.91 1197.61 125.35 10.47% 99.80%Day 14 949.01 1167.07 1117.96 1078.01 114.39 10.61% 89.83% Day 211276.91 1348.99 1005.55 1210.48 181.10 14.96% 100.87% Day 31 1025.13983.58 1238.35 1082.35 136.69 12.63% 90.20%

Example 10: Interference Study

Acceptability criteria: The difference due to a potential interferingsubstance should be ≤2SD or 20% CV to be considered acceptable.

Hemolysis Interference: Six spike pools were analyzed in triplicate fora baseline, slight, moderate and gross hemolysis interference.

Beta Amyloid 40 and Beta Amyloid 42 is only acceptable for non-hemolyzedand slightly hemolyzed cerebrospinal fluid samples.

Beta Amyloid 40 Pool 1 2000 pg/mL Hemolysis Baseline Slight ModerateGross Total Replicate 1 1844.27 2218.98 1972.21 2509.92 2136.35Replicate 2 1676.71 2156.41 2121.93 2030.17 1996.31 Replicate 3 1682.641930.54 1873.38 2301.92 1947.12 MEAN 1734.54 2101.98 1989.17 2280.672026.59 STDEV  95.08 151.73 125.14 240.58 153.13 % CV  5.48%  7.22% 6.29%  10.55%  7.38% % Recovery 86.73% 105.10% 99.46% 114.03% 101.33%Beta Amyloid 42 Pool 1 2000 pg/mL Hemolysis Baseline Slight ModerateGross Total Replicate 1 1710.73 2213.31 2587.34 2803 2328.60 Replicate 21608.45 1983.62 2322.18 2327.79 2060.51 Replicate 3 1652.88 2092.951827.26 3203.29 2194.10 MEAN 1657.35 2096.63 2245.59 2778.03 2194.40STDEV 51.29 114.89 385.78 438.28 247.56 % CV  3.09%  5.48%  17.18% 15.78%  10.38% % Recovery 82.87% 104.83% 112.28% 138.90% 109.72% BetaAmyloid 40 Pool 3 6000 pg/mL Hemolysis Baseline Slight Moderate GrossTotal Replicate 1 5243.65 5724.38 5902.54 5860.26 5682.71 Replicate 25677.02 5569.79 5947.92 5902.54 5774.32 Replicate 3 6116.8 5952.46660.26 5677.02 6101.62 MEAN 5679.16 5748.86 6170.24 5813.27 5852.88STDEV 436.58 192.48 424.98 119.88 293.48 % CV  7.69%  3.35%  6.89% 2.06%  5.00% % Recovery 94.65% 95.81% 102.84% 96.89% 97.55%

Beta Amyloid 42 Pool 3 6000 pg/mL Hemolysis Baseline Slight ModerateGross Total Replicate 1 4683.16 6010.68 7298 7298 6322.46 Replicate 25857.22 6187.23 7357.24 5857.22 6314.73 Replicate 3 6274.4 6725.445665.83 7858.76 6631.11 MEAN 5604.93 6307.78 6773.69 7004.66 6422.77STDEV 825.08 372.32 959.89 1032.51 797.45 % CV 14.72%  5.90%  14.17% 14.74%  12.38% % Recovery 93.42% 105.13% 112.89% 116.74% 107.05% BetaAmyloid 40 Pool 5 14000 pg/mL Hemolysis Baseline Slight Moderate GrossTotal Replicate 1 16318.69 16205.4 15909.32 17106.17 16384.90 Replicate2 15739.55 16847.72 16061.5 15891.29 16135.02 Replicate 3 17159.5116499.24 18690.45 15563.36 16978.14 MEAN 16405.92 16517.45 16887.0916186.94 16499.35 STDEV 713.99 321.55 1563.61 812.79 852.98 % CV  4.35% 1.95%  9.26%  5.02%  5.14% % Recovery 117.19% 117.98% 120.62% 115.62%117.85% Beta Amyloid 42 Pool 5 14000 pg/mL Hemolysis Baseline SlightModerate Gross Total Replicate 1 16275.91 14772.68 17788.89 15214.5916013.02 Replicate 2 16777.1 13701.79 16693.63 17433.68 16151.55Replicate 3 15508.59 16548.81 16234.96 15868.04 16040.10 MEAN 16187.2015007.76 16905.83 16172.10 16068.22 STDEV 638.89 1437.99 798.40 1140.361003.91 % CV  3.95%  9.58%  4.72%  7.05%  6.33% % Recovery 115.62%107.20% 120.76% 115.52% 114.77%

Beta Amyloid 40 Pool 2 4000 pg/mL Hemolysis Baseline Slight ModerateGross Total Replicate 1 3762.71 3785.66 4510.79 4256.2 4078.84 Replicate2 4562.35 4864.24 4423.28 4808.1 4664.49 Replicate 3 4679.51 4659.035073.88 3987.41 4599.96 MEAN 4334.86 4436.31 4669.32 4350.57 4447.76STDEV 498.94 572.75 353.08 418.40 460.79 % CV  11.51%  12.91%  7.56% 9.62%  10.40% % Recovery 108.37% 110.91% 116.73% 108.76% 111.19% BetaAmyloid 42 Pool 2 4000 pg/mL Baseline Slight Moderate Gross TotalReplicate 1 3589.03 3620.91 5005.48 4908.61 4281.01 Replicate 2 4496.675052.12 5638.52 5571.62 5189.73 Replicate 3 4112.79 4726.31 5032.285086.1 4739.37 MEAN 4066.16 4466.45 5225.43 5188.78 4736.70 STDEV 455.61750.16 358.00 343.22 476.75 % CV  11.20%  16.80%  6.85%  6.61%  10.37% %Recovery 101.65% 111.66% 130.64% 129.72% 118.42% Beta Amyloid 40 Pool 412000 pg/mL Hemolysis Baseline Slight Moderate Gross Total Replicate 113548.97 11514.4 10243.99 12982.46 12072.46 Replicate 2 10931.8511070.63 12083.48 12024.58 11527.64 Replicate 3 12232.13 11732.8810797.77 11122.15 11471.23 MEAN 12237.65 11439.30 11041.75 12043.0611690.44 STDEV 1308.57 337.45 943.70 930.29 880.00 % CV  10.69%  2.95% 8.55%  7.72%  7.48% % Recovery 101.98% 95.33% 92.01% 100.36% 97.42%

Beta Amyloid 42 Pool 4 12000 pg/mL Hemolysis Baseline Slight ModerateGross Total Replicate 1 12895.5 10962.68 11010.98 12132.09 11750.31Replicate 2 13332.29 10058.52 12917.13 11115.89 11855.96 Replicate 313337.19 13511.14 9223.21 13398.17 12367.43 MEAN 13188.33 11510.7811050.44 12215.38 11991.23 STDEV 253.61 1790.38 1847.28 1143.42 1258.67% CV  1.92% 15.55% 16.72%  9.36% 10.89% % Recovery 109.90% 95.92% 92.09%101.79% 99.93% Beta Amyloid 40 Pool 6 16000 pg/mL Hemolysis BaselineSlight Moderate Gross Total Replicate 1 16850.04 22436.41 19389.6918606.33 19320.62 Replicate 2 18143.58 20584.49 19475.64 18724.2619231.99 Replicate 3 18419.48 21353.42 19913.02 18236.68 19480.65 MEAN17804.37 21458.11 19592.78 18522.42 19344.42 STDEV 837.90 930.39 280.64254.39 575.83 % CV  4.71%  4.34%  1.43%  1.37%  2.96% % Recovery 111.28%134.11% 122.45% 115.77% 120.90% Beta Amyloid 42 Pool 6 16000 pg/mLHemolysis Baseline Slight Moderate Gross Total Replicate 1 15090.217802.59 23536.65 16841.59 18317.76 Replicate 2 16245.91 17194.8919780.66 17529.35 17687.70 Replicate 3 17331.23 16649.26 22671.9417116.46 18442.22 MEAN 16222.45 17215.58 21996.42 17162.47 18149.23STDEV 1120.70 576.94 1967.01 346.18 1002.71 % CV  6.91%  3.35%  8.94% 2.02%  5.30% % Recovery 101.39% 107.60% 137.48% 107.27% 113.43%

Lipemia Interference: Six spike pools were analyzed in triplicate for abaseline, slight, moderate and gross lipemic interference.

Beta Amyloid 40 and Beta Amyloid 42 is acceptable for slightly andmoderately lipemic cerebrospinal fluid samples.

Beta Amyloid 40 Pool 1 2000 pg/mL Lipemic Baseline Slight Moderate GrossTotal Replicate 1 2122.57 1867.23 1749.50 2130.24 1967.39 Replicate 21840.46 1975.60 1860.85 2158.83 1958.94 Replicate 3 1842.49 2105.691665.35 1960.04 1893.39 MEAN 1935.17 1982.84 1758.57 2083.04 1939.90STDEV 162.29 119.39 98.06 107.47 121.81 % CV  8.39%  6.02%  5.58%  5.16% 6.29% % Recovery 96.76% 99.14% 87.93% 104.15% 97.00% Beta Amyloid 40Pool 2 4000 pg/mL Lipemic Baseline Slight Moderate Gross Total Replicate1 3686.74 3982.2 3818.81 3567.06 3763.70 Replicate 2 4309.05 4176.513668.16 3896.53 4012.56 Replicate 3 3483.79 4223.27 3672.9 4053.583858.39 MEAN 3826.53 4127.33 3719.96 3839.06 3878.22 STDEV 430.02 127.8485.64 248.30 222.95 % CV 11.24%  3.10%  2.30%  6.47%  5.78% % Recovery95.66% 103.18% 93.00% 95.98% 96.96% Beta Amyloid 40 Pool 3 6000 pg/mLLipemic Baseline Slight Moderate Gross Total Replicate 1 6048.48 6028.485897.49 4912.21 5721.67 Replicate 2 5483.52 6205.15 6136.62 5144.545742.46 Replicate 3 5405.7 5779.68 5634.34 5051.26 5467.75 MEAN 5645.906004.44 5889.48 5036.00 5643.96 STDEV 350.81 213.75 251.24 116.91 233.18% CV  6.21%  3.56%  4.27%  2.32%  4.09% % Recovery 94.10% 100.07% 98.16%83.93% 94.07%

Beta Amyloid 40 Pool 4 12000 pg/mL Lipemic Baseline Slight ModerateGross Total Replicate 1 11795.73 12524.54 10057.38 10551.66 11232.33Replicate 2 11815.14 11947.41 11263.66 11027.67 11513.47 Replicate 312548.85 11322.75 10836.52 10965.53 11418.41 MEAN 12053.24 11931.5710719.19 10848.29 11388.07 STDEV 429.32 601.05 611.64 258.76 475.19 % CV 3.56%  5.04%  5.71%  2.39%  4.17% % Recovery 100.44% 99.43% 89.33%90.40% 94.90% Beta Amyloid 40 Pool 5 14000 pg/mL Lipemic Baseline SlightModerate Gross Total Replicate 1 13705.99 15174.54 15620.23 15814.315078.77 Replicate 2 14636.59 16544.85 14053.01 14138.08 14843.13Replicate 3 13981.06 14130.05 15965.99 14285.88 14590.75 MEAN 14107.8815283.15 15213.08 14746.09 14837.55 STDEV 478.09 1211.06 1019.41 928.05909.15 % CV  3.39%  7.92%  6.70%  6.29%  6.08% % Recovery 100.77%109.17% 108.66% 105.33% 105.98% Beta Amyloid 40 Pool 6 16000 pg/mLLipemic Baseline Slight Moderate Gross Total Replicate 1 13138.6915864.69 16090.04 15407.11 15125.13 Replicate 2 15532.57 14726.3315822.47 15995.82 15519.30 Replicate 3 14445.5 15980.5 15573.06 16628.4415656.88 MEAN 14372.25 15523.84 15828.52 16010.46 15433.77 STDEV 1198.62693.09 258.54 610.80 690.26 % CV  8.34%  4.46%  1.63%  3.81%  4.56% %Recovery 89.83% 97.02% 98.93% 100.07% 96.46%

Beta Amyloid 42 Pool 1 2000 pg/mL Lipemic Baseline Slight Moderate GrossTotal Replicate 1 1770.53 2216.73 1645.46 2049.87 1920.65 Replicate 21910.30 1882.26 1523.80 1956.37 1818.18 Replicate 3 1702.76 1946.211753.75 1931.18 1833.48 MEAN 1794.53 2015.07 1641.00 1979.14 1857.44STDEV 105.83 177.55 115.04 62.54 115.24 % CV  5.90%  8.81%  7.01%  3.16% 6.22% % Recovery 89.73% 100.75% 82.05% 98.96% 92.87% Beta Amyloid 42Pool 2 4000 pg/mL Lipemic Baseline Slight Moderate Gross Total Replicate1 3478.11 3586.59 4288.67 3411.32 3691.17 Replicate 2 4226.72 3458.623391.05 3641 3679.35 Replicate 3 3573.75 3530.16 3387.5 3892.18 3595.90MEAN 3759.53 3525.12 3689.07 3648.17 3655.47 STDEV 407.42 64.13 519.27240.51 307.83 % CV 10.84%  1.82% 14.08%  6.59%  8.33% % Recovery 93.99%88.13% 92.23% 91.20% 91.39% Beta Amyloid 42 Pool 3 6000 pg/mL LipemicBaseline Slight Moderate Gross Total Replicate 1 5663.22 4897.39 6049.814046.59 5164.25 Replicate 2 4662.02 5540.19 5727.69 4578.2 5127.03Replicate 3 4620.36 5042 4901.74 4681.98 4811.52 MEAN 4981.87 5159.865559.75 4435.59 5034.27 STDEV 590.44 337.22 592.17 340.86 465.17 % CV11.85%  6.54% 10.65%  7.68%  9.18% % Recovery 83.03% 86.00% 92.66%73.93% 83.90%

Beta Amyloid 42 Pool 4 pg/mL Lipemic Baseline Slight Moderate GrossTotal Replicate 1 10620.61 9407.96 8905.02 11148.72 10020.58 Replicate 210451.87 10683.08 11029.04 10561.05 10681.26 Replicate 3 10432.1610691.1 10686.19 11870.08 10919.88 MEAN 10501.55 10260.71 10206.7511193.28 10540.57 STDEV 103.58 738.52 1140.29 655.65 659.51 % CV  0.99% 7.20% 11.17%  5.86%  6.30% % Recovery 87.51% 85.51% 85.06% 93.28%87.84% Beta Amyloid 42 Pool 5 pg/mL Lipemic Baseline Slight ModerateGross Total Replicate 1 15585.98 15188.42 15258.95 15313.05 15336.60Replicate 2 14627.03 16766.84 14630.3 15586.4 15402.64 Replicate 314494.58 13756.62 17081.41 15074.36 15101.74 MEAN 14902.53 15237.2915656.89 15324.60 15280.33 STDEV 595.58 1505.71 1273.09 256.22 907.65 %CV  4.00%  9.88%  8.13%  1.67%  5.92% % Recovery 106.45% 108.84% 111.83%109.46% 109.15% Beta Amyloid 42 Pool 6 16000 pg/mL Lipemic BaselineSlight Moderate Gross Total Replicate 1 14926.96 14530.24 16083.9713096.47 14659.41 Replicate 2 17968.9 14741.02 14978.74 12618.8615076.88 Replicate 3 14277.43 16397.86 14946.5 13619.23 14810.26 MEAN15724.43 15223.04 15336.40 13111.52 14848.85 STDEV 1970.71 1022.87647.61 500.35 1035.39 % CV 12.53%  6.72%  4.22%  3.82%  6.82% % Recovery98.28% 95.14% 95.85% 81.95% 92.81%

Bilirubin Interference: Six spiked pools were analyzed in triplicate fora baseline, slight, moderate and gross icteric interference.

Beta Amyloid 40 and Beta Amyloid 42 are acceptable for non-icteric andslightly icteric cerebrospinal fluid samples.

Beta Amyloid 42 Pool 1 2000 pg/mL Icteric Baseline Slight Moderate GrossTotal Replicate 1 1488.32 1620.2 1654.67 1631.02 1598.55 Replicate 21910.86 1730.53 1531.75 1433.03 1651.54 Replicate 3 1883.89 1992.471675.22 1325.76 1719.34 MEAN 1761.02 1781.07 1620.55 1463.27 1656.48STDEV 236.55 191.21 77.58 154.86 165.05 % CV 13.43% 10.74%  4.79% 10.58% 9.88% % Recovery 88.05% 89.05% 81.03% 73.16% 82.82% Beta Amyloid 40Pool 1 2000 pg/mL Icteric Baseline Slight Moderate Gross Total Replicate1 2028.09 1870.09 1943.1 2162.25 2000.88 Replicate 2 2065.04 1909.072169.68 2054.9 2049.67 Replicate 3 1916.17 2030.58 2128.03 2144.22054.75 MEAN 2003.10 1936.58 2080.27 2120.45 2035.10 STDEV 77.52 83.71120.60 57.48 84.83 % CV  3.87%  4.32%  5.80%  2.71%  4.18% % Recovery100.16% 96.83% 104.01% 106.02% 101.76% Beta Amyloid 40 Pool 2 4000 pg/mLIcteric Baseline Slight Moderate Gross Total Replicate 1 4386.17 3259.783670.62 4098.44 3853.75 Replicate 2 3931.17 3534.09 3998.32 4156.483905.02 Replicate 3 4190.26 3432.95 3857.11 4144.8 3906.28 MEAN 4169.203408.94 3842.02 4133.24 3888.35 STDEV 228.23 138.72 164.37 30.70 140.51% CV  5.47%  4.07%  4.28%  0.74%  3.64% % Recovery 104.23% 85.22% 96.05%103.33% 97.21% Beta Amyloid 40 Pool 3 6000 pg/mL Icteric Baseline SlightModerate Gross Total Replicate 1 6020.23 5940.6 6532.72 5627.95 6030.38Replicate 2 6100.69 6203.06 6753.67 6142.42 6299.96 Replicate 3 5896.997228.16 6686.13 5976.64 6446.98 MEAN 6005.97 6457.27 6657.51 5915.676259.11 STDEV 102.60 680.38 113.22 262.60 289.70 % CV  1.71%  10.54% 1.70%  4.44%  4.60% % Recovery 100.10% 107.62% 110.96% 98.59% 104.32%

Beta Amyloid 40 Pool 4 12000 pg/mL Icteric Baseline Slight ModerateGross Total Replicate 1 12495.31 11656.12 11097.61 9822.68 11267.93Replicate 2 12830.89 11106.69 10397.3 9836.78 11042.92 Replicate 311888.74 12262.26 11055.11 11149.72 11588.96 MEAN 12404.98 11675.0210850.01 10269.73 11299.93 STDEV 477.53 578.02 392.63 762.13 552.58 % CV 3.85%  4.95%  3.62%  7.42%  4.96% % Recovery 103.37% 97.29% 90.42%85.58% 94.17% Beta Amyloid 40 Pool 5 14000 pg/mL Icteric Baseline SlightModerate Gross Total Replicate 1 11830.45 15117.44 13241.28 14397.7613646.73 Replicate 2 12060.76 15861.95 12366.55 15603.39 13973.16Replicate 3 12207.86 13692.37 14664.53 16441.16 14251.48 MEAN 12033.0214890.59 13424.12 15480.77 13957.13 STDEV 190.23 1102.44 1159.85 1027.20869.93 % CV  1.58%  7.40%  8.64%  6.64%  6.06% % Recovery 85.95% 106.36%95.89% 110.58% 99.69% Beta Amyloid 40 Pool 6 16000 pg/mL IctericBaseline Slight Moderate Gross Total Replicate 1 16532.34 12984.5314162.06 17720.56 15349.87 Replicate 2 14859.48 13685.93 16447.3117366.89 15589.90 Replicate 3 14600.09 15161.21 16017.61 16461.915560.20 MEAN 15330.64 13943.89 15542.33 17183.12 15499.99 STDEV 1048.761111.03 1214.50 649.14 1005.86 % CV  6.84%  7.97%  7.81%  3.78%  6.60% %Recovery 95.82% 87.15% 97.14% 107.39% 96.87%

Beta Amyloid 42 Pool 1 2000 pg/mL Icteric Baseline Slight Moderate GrossTotal Replicate 1 1488.32 1620.2 1654.67 1631.02 1598.55 Replicate 21910.86 1730.53 1531.75 1433.03 1651.54 Replicate 3 1883.89 1992.471675.22 1325.76 1719.34 MEAN 1761.02 1781.07 1620.55 1463.27 1656.48STDEV 236.55 191.21 77.58 154.86 165.05 % CV 13.43% 10.74%  4.79% 10.58% 9.88% % Recovery 88.05% 89.05% 81.03% 73.16% 82.82% Beta Amyloid 42Pool 2 4000 pg/mL Icteric Baseline Slight Moderate Gross Total Replicate1 3222.05 3732.25 3145.28 2994.84 3273.61 Replicate 2 3497.19 3341.183648.74 3014.42 3375.38 Replicate 3 3611.9 3832.09 3394.39 2847.733421.53 MEAN 3443.71 3635.17 3396.14 2952.33 3356.84 STDEV 200.35 259.45251.73 91.11 200.66 % CV  5.82%  7.14%  7.41%  3.09%  5.86% % Recovery86.09% 90.88% 84.90% 73.81% 83.92% Beta Amyloid 42 Pool 3 6000 pg/mLIcteric Baseline Slight Moderate Gross Total Replicate 1 5144.23 4728.274239.65 3822.39 4483.64 Replicate 2 5114.94 5040.95 4875.55 3903.054733.62 Replicate 3 4701.39 4930.46 4063.35 3774.87 4367.52 MEAN 4986.854899.89 4392.85 3833.44 4528.26 STDEV 247.65 158.57 427.22 64.80 224.56% CV  4.97%  3.24%  9.73%  1.69%  4.90% % Recovery 83.11% 81.66% 73.21%63.89% 75.47%

Beta Amyloid 42 Pool 4 12000 pg/mL Icteric Baseline Slight ModerateGross Total Replicate 1 10827.22 10391.44 10703.95 12734.98 11164.40Replicate 2 10500.06 9871.15 11285.4 10390.82 10511.86 Replicate 310391.44 10156.67 10308.04 14883.95 11435.03 MEAN 10572.91 10139.7510765.80 12669.92 11037.09 STDEV 226.84 260.56 491.61 2247.27 806.57 %CV  2.15%  2.57%  4.57%  17.74%  6.75% % Recovery 88.11% 84.50% 89.71%105.58% 91.98% Beta Amyloid 42 Pool 5 14000 pg/mL Icteric BaselineSlight Moderate Gross Total Replicate 1 14597.66 15694.71 16331.0216887.67 15877.77 Replicate 2 14635.85 15134.01 16958.66 15572.3415575.22 Replicate 3 14213.69 16612.21 20720.11 16993.48 17134.87 MEAN14482.40 15813.64 18003.26 16484.50 16195.95 STDEV 233.49 746.24 2373.69791.72 1036.29 % CV  1.61%  4.72%  13.18%  4.80%  6.08% % Recovery103.45% 112.95% 128.59% 117.75% 115.69% Beta Amyloid 42 Pool 6 16000pg/mL Icteric Baseline Slight Moderate Gross Total Replicate 1 14827.9416055.37 16949.58 20372.18 17051.27 Replicate 2 17956.96 18569.8116515.45 18956.15 17999.59 Replicate 3 20671.44 19288.36 17146.7718702.2 18952.19 MEAN 17818.78 17971.18 16870.60 19343.51 18001.02 STDEV2924.20 1697.59 322.99 899.86 1461.16 % CV  16.41%  9.45%  1.91%  4.65% 8.11% % Recovery 111.37% 112.32% 105.44% 120.90% 112.51%

Example 11: Ion Suppression

Ten patient samples were extracted.

The acquisition window was opened up to 10 minutes to monitor ionsuppression across the gradient. The ten samples were injected throughthe analytical column while the digested peptide mix of beta amyloid 40and 42 were infused post-column

If the total ion chromatogram (TIC) of AB40 or AB42 showed a decrease of≥15% of signal intensity when the internal standards for AB40 or AB42eluted then ion suppression would be determined to be present in theassay.

The TIC of the digested peptides of AB40 and 42 showed no suppression inthe gradient when the analyte is eluting. The TIC signal intensity is aflat line and shows ≤15% difference in signal intensity which is withinthe acceptable parameters of the assay.

Example 12: Carryover

High calibrator standards analyzed followed by four matrix blanks, thissequence was repeated another two times. The mean calculatedconcentration of the matrix blanks after the high calibrator yields a %Recovery of 0.06% for both Beta Amyloid 40 and 42.

There is no carryover observed for this assay.

pg/mL AB 42 AB 40 High_std_1 24114.04 30552.94 High_std_2 21837.2527101.48 High_std_3 23955.83 26691.95 High_std_4 24661.76 26510.1Blank_1 3.20 22.85 Blank_2 0.05 16.97 Blank_3 15.31 14.62 Blank_4 19.3437.13 High_std_5 23467.62 29809.18 High_std_6 21763.07 26340.7High_std_7 21486.03 26665.22 High_std_8 22325.03 26431.58 Blank_5 11.10−1.1 Blank_6 5.28 50.1 Blank_7 15.80 9.81 High_std_18 23019.97 25465.63High_std_19 19756.77 24195.08 High_std_20 26221.01 26983.93 High_std_2126237.81 27483.79 Blank_15 14.56 14.64 Blank_16 12.74 10.31 Blank_1719.97 5.05 Blank_18 25.07 1.96 Blank High Standard % Recovery AB42 MeanCalculated Value 12.95 23237.18 0.06% Mean Standard Deviation 7.601941.57 AB40 Mean Calculated Value 16.58 27019.30 0.06% Mean StandardDeviation 15.29 1710.44

Example 13: Reference Interval (RI)

Beta Amyloid 40: 6000.00-15000.00 pg/mL

Beta Amyloid 42: 700.00-4000.00 pg/mL

Example 14 Alzheimer's Patient Data

Cerebrospinal fluids (CSF) of 211 subjects, which include patientsdiagnosed with Alzheimer's disease and normal subjects, were analyzed.FIGS. 8-10.

Aβ40 and Aβ42 were detected in all CSF samples. Surprisingly, Aβ42levels were about 10 times higher than what has been published. Majorityof patient samples were in the range of 1 to 8 ng/mL Alzheimer'spatients were distinguishable based on the low ratio of Aβ42:Aβ40, ascompared to the borderline patients and normal subjects (highest ratio).FIG. 8.

Example 15: Additional Recovery Study

Sample Preparation: All plastic disposables were pretreated, and Aβstandards were stabilized to prevent nonspecific binding and to enhancelong-term storage stability.

A strong protein denaturant was added to human CSF (500 uL), and samplesunderwent a protein digestion followed by solid-phase extraction.Samples were processed on a robotic liquid handler (Hamilton MicrolabStar A857) in conjunction with a CEREX IP8 (SPEware) solid-phaseextraction manifold.

Separation: HPLC separation was performed on an Aria TLX-4 System(Thermo Scientific) using a Waters XBridge Protein BEH C4 Column,4.6×100 mm, 3.5 micron 300 Å

Detection: Thermo TSQ Quantiva Triple Quadrupole Mass Spectrometer

Linearity data show an R² value of at least 0.98 for both peptides, witha % CV of ≤15% across 8 calibrator standards.

The limit of quantitation was 100 pg/mL for both peptides.

Assay precision (% CV) was ≤15%, with a recovery range of 84% to 112%for Aβ40 and Aβ42.

Intra-assay (N=10) and Inter-assay (N=5, 5 days) Precision and Accuracy

Quality Expected Intra-assay Inter-assay Control Concentration MeanAccuracy Mean Accuracy Peptide Level (pg/mL) (pg/mL) (%) CV (%) (pg/mL)(%) CV (%) Aβ40 Low 750 643 86 14 800 107 15 Med 7,500 8,050 107 128,127 108 9 High 15,000 16,174 108 7 16,597 111 8 Aβ42 Low 750 656 87 10722 96 15 Med 7,500 7,363 98 6 6,997 93 6 High 15,000 14,614 97 4 14,83699 8

Assay Stability Over 8 months

Quality Expected October 2015 December 2015 June 2016 ControlConcentration Measured Accuracy Measured Accuracy Measured AccuracyPeptide Level (pg/mL) (pg/mL) (%) (pg/mL) (%) (pg/mL) (%) Aβ40 Low 750660 88 704 94 758 101 Med 7,500 6,891 92 7,608 101 6,987 93 High 15,00012,656 84 15,426 103 14,055 94 Aβ42 Low 750 704 94 820 109 815 109 Med7,500 8,381 112 8,074 108 6,596 88 High 15,000 13,037 87 16,853 11214,362 96

Stabilization of Aβ peptides was achieved, allowing for long-termstorage of calibrator and quality control standards.

Frozen calibrators have been stable for at least 8 months when stored at−80° C.

Stabilization measures eliminated nonspecific binding and yielded ahigher analyte recovery. FIGS. 12 and 13.

Values for patient CSF Aβ42 concentrations were higher using theLC-MS/MS assay than those using ELISA assays. This is consistent withprevious reports comparing the 2 methodolgies. Future work willdetermine if the pre-analytical factors described herein might helpexplain the discrepancy.

LC-MS/MS ELISA Patient (pg/mL) (pg/mL) 1 1,668 536 2 1,617 534 3 2,808624 4 3,184 850 5 4,061 913 6 2,142 672 7 1,878 627 8 2,829 566 9 1,882633 10 1,634 614 11 2,706 644 12 773 466 13 1,801 602 14 3,891 739 151,524 671 16 2,300 605 17 1,167 458 18 1,842 625 19 2,431 665 20 2,814938

Conclusion: This novel approach eliminated one of the main challenges inquantitating Aβ40 and Aβ42: poor reproducibility caused by nonspecificbinding.

Example 16: Patient Diagnostic Study

Three quality controls (low, medium, high) were ran representing threedistinct points along the calibration curve. QC's were ran at thebeginning and end of the run to ensure accurate quantitation throughoutthe plate. Quality control accuracy is shown below:

Abeta 40 Expected(pg/mL) Calculated(pg/mL) % Accuracy Front: Low QC 750743.28  99.10% Front: Medium QC 7500 6724.33  89.66% Front: High QC15000 13554.62  90.36% Back: Low QC 750 754.58 100.61% Back: Medium QC7500 7351.87  98.02% Back: High QC 15000 13911.65  92.74%

Expected Calculated Abeta 42 (pg/mL) (pg/mL) % Accuracy Front: Low QC750 815.71 108.76% Front: Medium QC 7500 6930.69  92.41% Front: High QC15000 13976.57  93.18% Back: Low QC 750 667.22  88.96% Back: Medium QC7500 6341.3  84.55% Back: High QC 15000 13384.45  89.23%

Aβ40 and 42 Patient Samples Ranges

Based off of 72 patient samples

Aβ40: 5135.33-25348.94 pg/mL

Aβ42: 1068.00-5499.76 pg/mL

Normalize varying levels of Aβ40 and 42.

As Aβ42 values decrease due to plaqueing or insufficient clearance, theratio of Aβ42/40 also decreases.

Aβ40 and 42 levels appear to increase/decrease independent of oneanother.

Data was sorted by Aβ42/40 ratio and split into two sets based of offthe median (Aβ42/40=0.17)

Half of the data into <Aβ42/40 Median

Half of the data into >Aβ42/40 Median

Mean, standard deviation, and % CV was calculated:

For ratio values <Aβ42/40 Median, values ≥1SD were removed

For ratio values >Aβ42/40 Median, values ≤1SD were removed

1SD outliers were placed into a “Borderline” category

The contents of the articles, patents, and patent applications, and allother documents and electronically available information mentioned orcited herein, are hereby incorporated by reference in their entirety tothe same extent as if each individual publication was specifically andindividually indicated to be incorporated by reference. Applicantsreserve the right to physically incorporate into this application anyand all materials and information from any such articles, patents,patent applications, or other physical and electronic documents.

The methods illustratively described herein may suitably be practiced inthe absence of any element or elements, limitation or limitations, notspecifically disclosed herein. Thus, for example, the terms“comprising”, “including,” containing”, etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof. It is recognized that various modifications arepossible within the scope of the invention claimed. Thus, it should beunderstood that although the present invention has been specificallydisclosed by preferred embodiments and optional features, modificationand variation of the invention embodied therein herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this invention.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the methods. This includes the genericdescription of the methods with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

Other embodiments are within the following claims. In addition, wherefeatures or aspects of the methods are described in terms of Markushgroups, those skilled in the art will recognize that the invention isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group.

That which is claimed is:
 1. A method for determining the amount ofamyloid beta fragments in a sample, said method comprising: (a)digesting amyloid beta in the sample to generate amyloid beta 40 (Aβ40)comprising the sequence GAIIGLMVGGVV (SEQ ID NO:2) and amyloid beta 42(Aβ42) comprising the sequence GAIIGLMVGGVVIA (SEQ ID NO:4); (b)purifying Aβ40 and Aβ42 in the sample; (b) ionizing Aβ40 and Aβ42 in thesample to produce one or more precursor ions; (c) generating one or morefragment ions of Aβ40 and Aβ42; and (d) determining the amount of theone or more fragment ion(s) from step (d) by mass spectrometry, whereinthe one or more fragment ions comprises an ion with a mass/charge ratioselected from the group consisting of 812.37±0.5, 869.4±0.5, 968.43±0.5,869.39±0.5, 968.44±0.5, 1067.5±0.5, and 1180.57±0.5.
 2. The method ofclaim 1, wherein said purifying comprises liquid chromatography.
 3. Themethod of claim 2, wherein said liquid chromatography comprises highperformance liquid chromatography (HPLC).
 4. The method of claim 1,wherein said method further comprises a C-4 analytical column
 5. Themethod of claim 1, further comprising pretreating surfaces of equipmentthat come in contact with the sample with an agent that prevents amyloidbeta from sticking to the surfaces.
 6. The method of claim 5, whereinthe agent is E. coli lysate.
 7. The method of claim 1, furthercomprising incubating the sample with an agent that stabilizes amyloidbeta.
 8. The method of claim 7, wherein said agent comprises an antibodythat binds to the C-terminus of amyloid beta, an antibody that binds tothe N-terminus of amyloid beta, apolipoprotein E2, apolipoprotein E4, ora combination thereof.
 9. The method of claim 7, wherein said agentconfers stability through at least three freeze-thaw cycles.
 10. Themethod of claim 7, wherein said agent confers stability for at least 2months at −70° C.
 11. The method of claim 1, wherein the methodcomprises a mixed mode anion exchange extraction.
 12. The method ofclaim 1, wherein said ionization comprises heated electrosprayionization (HESI).
 13. The method of claim 1, wherein said ionizationcomprises ionizing in positive mode.
 14. The method of claim 1, whereinsaid generation of fragment ions comprises using collision energy ofbetween 20V to 45V.
 15. The method of claim 1, further comprising addingan internal standard.
 16. The method of claim 15, wherein said internalstandard is isotopically labeled.
 17. The method of claim 16, whereinsaid internal standard comprises ¹³C¹⁵N labeling.
 18. The method ofclaim 17, wherein the precursor ion of the internal standard has amass/charge ratio of 1110.7±0.5.
 19. The method of claim 17, wherein theone or more fragment ions of the internal standard has a mass/chargeratio of 768.48±0.5, 825.5±0.5, or 882.52±0.5.
 20. The method of claim1, wherein the limit of quantitation of the method is less than or equalto 10 ng/mL.
 21. The method of claim 1, wherein said digestion comprisesdigesting with Lys C.
 22. The method of claim 21, wherein said digestionfurther comprises adding urea.
 23. The method of claim 22, wherein saiddigestion further comprises digestion in microwave.
 24. The method ofclaim 1, wherein the amyloid beta fragment further comprises a wingedpeptide.
 25. The method of claim 24, wherein the winged peptide ishydrophilic.
 26. The method of claim 24, wherein the winged peptidecomprises one or more N-terminal or C-terminal amino acid residues. 27.The method of claim 1, wherein the method comprises determining theratio of Aβ42 to Aβ40.